Thyroid hormone analogs and methods of use

ABSTRACT

Disclosed are methods of treating subjects having conditions related to angiogenesis including administering an effective amount of a polymeric form of thyroid hormone, or an antagonist thereof, to promote or inhibit angiogenesis in the subject. Compositions of the polymeric forms of thyroid hormone, or thyroid hormone analogs, are also disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/975,735 entitled THYROID HORMONE ANALOGS AND METHODS OF USEIN ANGIOGENESIS, filed Aug. 26, 2013, which is a continuation-in-part ofU.S. patent application Ser. No. 12/626,068, filed Nov. 25, 2009, whichis a divisional application of U.S. Pat. No. 7,785,632 filed Sep. 15,2004, which claims priority to U.S. Patent Application No. 60/502,721,filed Sep. 15, 2003, the content of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to thyroid hormone, thyroid hormone analogs andderivatives, and polymeric forms thereof. Methods of using suchcompounds and pharmaceutical compositions containing the same are alsodisclosed. The invention also relates to methods of preparing suchcompounds.

BACKGROUND OF THE INVENTION

Thyroid hormones, L-thyroxine (T4) and L-triiodothyronine (T3), regulatemany different physiological processes in different tissues invertebrates. Most of the actions of thyroid hormones are mediated by thethyroid hormone receptor (“TR”), which is a member of the nuclearreceptor superfamily of ligand-activated transcription regulators. Thissuperfamily also includes receptors for steroid hormones, retinoids, and1,25-dihydroxyvitamin D3. These receptors are transcription factors thatcan regulate expression of specific genes in various tissues and aretargets for widely used drugs, such as tamoxifen, an estrogen receptorpartial antagonist. There are two different genes that encode twodifferent TRs, TRα and TRβ. These two TRs are often co-expressed atdifferent levels in different tissues. Most thyroid hormones do notdiscriminate between the two TRs and bind both with similar affinities.

Gene knockout studies in mice indicate that TRβ plays a role in thedevelopment of the auditory system and in the negative feedback ofthyroid stimulating hormone by T3 in the pituitary, whereas TRαmodulates the effect of thyroid hormone on calorigenesis and on thecardiovascular system. The identification of TR antagonists could playan important role in the future treatment of hypothyroidism. Suchmolecules would act rapidly by directly antagonizing the effect ofthyroid hormone at the receptor level, a significant improvement forindividuals with hypothyroidism who require surgery, have cardiacdisease, or are at risk for life-threatening thyrotoxic storm.

Thus, there remains a need for the development of compounds thatselectively modulate thyroid hormone action by functioning asisoform-selective agonists or antagonists of the thyroid hormonereceptors (TRs). Recent efforts have focused on the design and synthesisof thyroid hormone (T3/T4) antagonists as potential therapeutic agentsand chemical probes. There is also a need for the development ofthyromimetic compounds that are more accessible than the natural hormoneand have potentially useful receptor binding and activation properties.

It is estimated that five million people are afflicted with chronicstable angina in the United States. Each year 200,000 people under theage of 65 die from what is termed “premature ischemic heart disease.”Despite medical therapy, many others go on to suffer myocardialinfarction and debilitating symptoms prompting the need forrevascularization with either percutaneous transluminal coronaryangioplasty or coronary artery bypass surgery. It has been postulatedthat one way of relieving myocardial ischemia would be to enhancecoronary collateral circulation.

Correlations have now been made between the anatomic appearance ofcoronary collateral vessels (“collaterals”) visualized at the time ofintracoronary thrombolytic therapy during the acute phase of myocardialinfarction and the creatine kinase time-activity curve, infarct size,and aneurysm formation. These studies demonstrate a protective role ofcollaterals in hearts with coronary obstructive disease, showing smallerinfarcts, less aneurysm formation, and improved ventricular functioncompared with patients in whom collaterals were not visualized. When thecardiac myocyte is rendered ischemic, collaterals develop actively bygrowth with DNA replication and mitosis of endothelial and smooth musclecells. Once ischemia develops, these factors are activated and becomeavailable for receptor occupation, which may initiate angiogenesis afterexposure to exogenous heparin. Unfortunately, the “natural” process bywhich angiogenesis occurs is inadequate to reverse the ischemia inalmost all patients with coronary artery disease.

During ischemia, adenosine is released through the breakdown of ATP.Adenosine participates in many cardio-protective biological events.Adenosine has a role in hemodynamic changes such as bradycardia andvasodilation, and has been suggested to have a role in such unrelatedphenomena as preconditioning and possibly the reduction in reperfusioninjury (Ely and Beme, Circulation, 85: 893 (1992).

Angiogenesis is the development of new blood vessels from preexistingblood vessels (Mousa, S. A., In Angiogenesis Inhibitors and Stimulators:Potential Therapeutic Implications, Landes Bioscience, Georgetown, Tex.;Chapter 1, (2000)). Physiologically, angiogenesis ensures properdevelopment of mature organisms, prepares the womb for egg implantation,and plays a key role in wound healing. The development of vascularnetworks during embryogenesis or normal and pathological angiogenesisdepends on growth factors and cellular interactions with theextracellular matrix (Breier et al., Trends in Cell Biology 6:454-456(1996); Folkman, Nature Medicine 1:27-31 (1995); Risau, Nature386:671-674 (1997). Blood vessels arise during embryogenesis by twoprocesses: vasculogenesis and angiogenesis (Blood et al., Bioch.Biophys. Acta 1032:89-118 (1990). Angiogenesis is a multi-step processcontrolled by the balance of pro- and anti-angiogenic factors. Thelatter stages of this process involve proliferation and the organizationof endothelial cells (EC) into tube-like structures. Growth factors suchas FGF2 and VEGF are thought to be key players in promoting endothelialcell growth and differentiation.

Control of angiogenesis is a complex process involving local release ofvascular growth factors (P Carmeliet, Ann NY Acad Sci 902:249-260,2000), extracellular matrix, adhesion molecules, and metabolic factors(R J Tomanek, G C Schatteman, Anat Rec 261:126-135, 2000). Mechanicalforces within blood vessels may also play a role (O Hudlicka, Molec CellBiochem 147:57-68, 1995). The principal classes of endogenous growthfactors implicated in new blood vessel growth are the fibroblast growthfactor (FGF) family and vascular endothelial growth factor (VEGF)(GPages, Ann NY Acad Sci 902:187-200, 2000). The mitogen-activated proteinkinase (MAPK; ERK1/2) signal transduction cascade is involved both inVEGF gene expression and in control of proliferation of vascularendothelial cells.

Intrinsic adenosine may facilitate the coronary flow response toincreased myocardial oxygen demands and so modulate the coronary flowreserve (Ethier et al., Am. J. Physiol., H131 (1993)) by demonstratingthat the addition of physiological concentrations of adenosine to humanumbilical vein endothelial cell cultures stimulates proliferation,possibly via a surface receptor. Adenosine may be a factor for humanendothelial cell growth and possibly angiogenesis. Angiogenesis appearsto be protective for patients with obstructive blood flow such ascoronary artery disease (“CAD”), but the rate at which blood vesselsgrow naturally is inadequate to reverse the disease. Thus, strategies toenhance and accelerate the body's natural angiogenesis potential shouldbe beneficial in patients with CAD.

Similarly, wound healing is a major problem in many developing countriesand diabetics have impaired wound healing and chronic inflammatorydisorders, with increased use of various cyclooxygenase-2 (COX2)inhibitors. Angiogenesis is necessary for wound repair since the newvessels provide nutrients to support the active cells, promotegranulation tissue formation, and facilitate the clearance of debris.Approximately 60% of the granulation tissue mass is composed of bloodvessels which also supply the necessary oxygen to stimulate repair andvessel growth. It is well documented that angiogenic factors are presentin wound fluid and promote repair while antiangiogenic factors inhibitrepair. Wound angiogenesis is a complex multi-step process. Despite adetailed knowledge about many angiogenic factors, little progress hasbeen made in defining the source of these factors, the regulatory eventsinvolved in wound angiogenesis, and in the clinical use of angiogenicstimulants to promote repair. Further complicating the understanding ofwound angiogenesis and repair is the fact that the mechanisms andmediators involved in repair likely vary depending on the depth of thewound, type of wound (burn, trauma, etc.), and the location (muscle,skin, bone, etc.). The condition and age of the patient (diabetic,paraplegic, on steroid therapy, elderly vs infant, etc) can alsodetermine the rate of repair and response to angiogenic factors. The sexof the patient and hormonal status (premenopausal, post menopausal,etc.) may also influence the repair mechanisms and responses. Impairedwound healing particularly affects the elderly and many of the 14million diabetics in the United States. Because reduced angiogenesis isoften a causative agent for wound healing problems in these patientpopulations, it is important to define the angiogenic factors importantin wound repair and to develop clinical uses to prevent and/or correctimpaired wound healing.

Thus, there remains a need for an effective therapy in the way ofangiogenic agents as either primary or adjunctive therapy for promotionof wound healing, coronary angiogenesis, or other angiogenic-relateddisorders, with minimum side effects. Such a therapy would beparticularly useful for patients who have vascular disorders such asmyocardial infarctions, stroke or peripheral artery diseases and couldbe used prophylactically in patients who have poor coronary circulation,which places them at high risk of ischemia and myocardial infarctions.

It is interesting to note that angiogenesis also occurs in othersituations, but which are undesirable, including solid tumor growth andmetastasis; rheumatoid arthritis; psoriasis; scleroderma; and threecommon causes of blindness—diabetic retinopathy, retrolentalfibroplasia, and neovascular glaucoma (in fact, diseases of the eye arealmost always accompanied by vascularization). The process of woundangiogenesis actually has many features in common with tumorangiogenesis. Thus, there are some conditions, such as diabeticretinopathy or the occurrence of primary or metastatic tumors, whereangiogenesis is undesirable. Thus, there remains a need for methods bywhich to inhibit the effect of angiogenic agents.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that thyroid hormone,thyroid hormone analogs, and their polymeric forms, act at the cellmembrane level and have pro-angiogenic properties that are independentof the nuclear thyroid hormone effects. Accordingly, these thyroidhormone analogs and polymeric forms (i.e., angiogenic agents) can beused to treat a variety of disorders. Similarly, the invention is alsobased on the discovery that thyroid hormone analog antagonists inhibitthe pro-angiogenic effect of such analogs, and can also be used to treata variety of disorders.

Accordingly, in one aspect the invention features methods for treating acondition amenable to treatment by inhibiting angiogenesis byadministering to a subject in need thereof an effective amount of acompound selected from the group consisting of tetriodothyroacetic acid(tetrac), triiodothyroacetic acid (triac) and a combination thereofconjugated via a covalent bond to a polymer, wherein said polymer ispolyglycolide, polylactic acid, or co-polymers thereof, wherein saidpolymer is formulated into a nanoparticle, wherein said nanoparticle isless than 200 nanometers, and wherein the administered compound acts atthe cell membrane level for inhibiting pro-angiogenesis agents.

In one embodiment, the thyroid hormone analog may be used to treat acondition wherein the condition amenable to treatment byanti-angiogenesis is a primary tumor, metastatic tumor, or diabeticretinopathy.

Thyroid hormone, thyroid hormone analogs, or polymeric forms thereofaccording to the invention can also be co-administered with one or morebiologically active substances that can include, for example, growthfactors, vasodilators, anti-coagulants, anti-virals, anti-bacterials,anti-inflammatories, immuno-suppressants, analgesics, vascularizingagents, or cell adhesion molecules, or combinations thereof. In oneembodiment, the thyroid hormone analog or polymeric form is administeredas a bolus injection prior to or post-administering one or morebiologically active substance.

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to, primary or metastatictumors, diabetic retinopathy, and related conditions. Examples of theanti-angiogenesis agents used for inhibiting angiogenesis are alsoprovided by the invention and include, but are not limited to,tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac),monoclonal antibody LM609, XT 199 or combinations thereof. Suchanti-angiogenesis agents can act at the cell surface to inhibit thepro-angiogenesis agents.

In one embodiment, the anti-angiogenesis agent is administered by aparenteral, oral, rectal, or topical mode, or combination thereof. Inanother embodiment, the anti-angiogenesis agent can be co-administeredwith one or more anti-angiogenesis therapies or chemotherapeutic agents.Chemotherapeutic agents may include, but are not limited to,doxorubicin, etoposide, cyclophophamide, 5-fluoracil, cisplatin,trichostatin A, paclitaxel, gemcitabine, taxotere, cisplatinum,carboplatinum, irinotecan, topotecan, adrimycin, bortezomib, and anycombinations or derivatives thereof.

The details of one or more embodiments of the invention have been setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural references unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: depicts the effects of T4 and T3 on angiogenesis quantitated inthe chick Chorioallantoic Membrane (CAM) assay. Control samples wereexposed to PBS and additional samples to 1 nM T3 or 0.1 μM T4 for 3days. Both hormones caused increased blood vessel branching in theserepresentative images from 3 experiments.

FIG. 1B: depicts the tabulation of mean±SEM of new branches formed fromexisting blood vessels during the experimental period drawn from 3experiments, each of which included 9 CAM assays. At the concentrationsshown, T3 and T4 caused similar effects (1.9-fold and 2.5-foldincreases, respectively, in branch formation). **P<0.001 by 1-way ANOVA,comparing hormone-treated with PBS-treated CAM samples.

FIG. 2A: depicts tetrac inhibiting stimulation of angiogenesis by T4,including 2.5-fold increase in blood vessel branch formation is seen ina representative CAM preparation exposed to 0.1 μM T4 for 3 days. In 3similar experiments, there was a 2.3-fold increase. This effect of thehormone is inhibited by tetrac (0.1 μM), a T4 analogue shown previouslyto inhibit plasma membrane actions of T4.

FIG. 2B: depicts T4-ag (0.1 μM) stimulating angiogenesis 2.3-fold(2.9-fold in 3 experiments), an effect also blocked by tetrac.

FIG. 2C: depicts a summary of the results of 3 experiments that examinethe actions of tetrac (which when administered alone does not stimulateangiogenesis), T4-ag, and T4 in the CAM assay. Data (means±SEM) wereobtained from 10 images for each experimental condition in each of 3experiments. **P<0.001 by ANOVA, comparing T4-treated andT4-agarose-treated samples with PBS-treated control samples.

FIG. 3A: depicts that the tandem effects of T4 (0.05 μM) and FGF2 (0.5μg/mL) in submaximal concentrations are additive in the CAM assay andequal the level of angiogenesis seen with FGF2 (1 μg/mL in the absenceof T4).

FIG. 3B: depicts a summary of results from 3 experiments that examinedactions of FGF2 and T4 in the CAM assay (means±SEM) as in A. *P<0.05;**P<0.001, comparing results of treated samples with those ofPBS-treated control samples in 3 experiments.

FIG. 4A: depicts the effects of anti-FGF2 on angiogenesis caused by T4or exogenous FGF2, wherein FGF2 caused a 2-fold increase in angiogenesisin the CAM model in 3 experiments, an effect inhibited by antibody (ab)to FGF2 (8 μg). T4 also stimulated angiogenesis 1.5-fold, and thiseffect was also blocked by FGF2 antibody, indicating that the action ofthyroid hormone in the CAM model is mediated by an autocrine/paracrineeffect of FGF2 because T4 and T3 cause FGF2 release from cells in theCAM model (Table 1). We have shown previously that a nonspecific IgGantibody has no effect on angiogenesis in the CAM assay.

FIG. 4B: depicts the summary of results from 3 CAM experiments thatstudied the action of FGF2-ab in the presence of FGF2 or T4. *P<0.01;**P<0.001, indicating significant effects in 3 experiments studying theeffects of thyroid hormone and FGF2 on angiogenesis and loss of theseeffects in the presence of antibody to FGF2.

FIG. 5A: depicts the effect of PD 98059, a MAPK (ERK1/2) signaltransduction cascade inhibitor, on angiogenesis induced by T4, T3, andFGF2 specifically, demonstrating that angiogenesis stimulated by T4 (0.1μM) and T3 (1 nM) together is fully inhibited by PD 98059 (3 μM).

FIG. 5B: depicts angiogenesis induced by FGF2 (1 μg/mL) is alsoinhibited by PD 98059, indicating that the action of the growth factoris also dependent on activation of the ERK1/2 pathway. In the context ofthe experiments involving T4-agarose (T4-ag) and tetrac (FIG. 2)indicating that T4 initiates its proangiogenic effect at the cellmembrane, results shown in FIGS. 5A and B are consistent with 2 rolesplayed by MAPK in the proangiogenic action of thyroid hormone: ERK1/2transduces the early signal of the hormone that leads to FGF2elaboration and transduces the subsequent action of FGF2 onangiogenesis.

FIG. 5C: depicts the summary of results of 3 experiments, represented byA and B, showing the effect of PD98059 on the actions of T4 and FGF2 inthe CAM model.

*P<0.01; **P<0.001, indicating results of ANOVA on data from 3experiments.

FIG. 6A: depicts that T4 and FGF2 activate MAPK in ECV304 endothelialcells. Cells were prepared in M199 medium with 0.25% hormone-depletedserum and treated with T4 (0.1 μM) for 15 minutes to 6 hours. Cells wereharvested and nuclear fractions were prepared. Nucleoproteins, separatedby gel electrophoresis, were immunoblotted with antibody tophosphorylated MAPK (pERK1 and pERK2, 44 and 42 kDa, respectively),followed by a second antibody linked to a luminescence-detection system.A β-actin immunoblot of nuclear fractions serves as a control for gelloading in each part of this figure. Each immunoblot is representativeof 3 experiments. T4 causes increased phosphorylation and nucleartranslocation of ERK1/2 in ECV304 cells. The effect is maximal in 30minutes, although the effect remains for >6 hours.

FIG. 6B: depicts ECV304 cells treated with the ERK1/2 activationinhibitor PD 98059 (PD; 30 μM) or the PKC inhibitor CGP41251 (CGP; 100nM) for 30 minutes, after which 10⁻⁷ M T4 was added for 15 minutes tocell samples as shown. Nuclei were harvested, and this representativeexperiment shows increased phosphorylation (activation) of ERK1/2 by T4(lane 4), which is blocked by both inhibitors (lanes 5 and 6),suggesting that PKC activity is a requisite for MAPK activation by T4 inendothelial cells.

FIG. 6C: depicts ECV304 cells treated with either T4 (10⁻⁷ mol/L), FGF2(10 ng/mL), or both agents for 15 minutes. The figure shows pERK1/2accumulation in nuclei with either hormone or growth factor treatmentand enhanced nuclear pERK1/2 accumulation with both agents together.

FIG. 7: depicts T4 increasing accumulation of FGF2 cDNA in ECV304endothelial cells. Cells were treated for 6 to 48 hours with T4 (10⁻⁷mol/L) and FGF2 and GAPDH cDNAs isolated from each cell aliquot. Thelevels of FGF2 cDNA, shown in the top blot, were corrected forvariations in GAPDH cDNA content, shown in the bottom blot, and thecorrected levels of FGF2 are illustrated below in the graph (mean±SE ofmean; n=2 experiments). There was increased abundance of FGF2 transcriptin RNA extracted from cells treated with T4 at all time points. *P<0.05;**P<0.01, indicating comparison by ANOVA of values at each time point tocontrol value.

FIG. 8: depicts an illustration of the CAM model of tumor implant of the7 day chick embryo tumor growth model

FIG. 9: depicts photographs of human dermal fibroblast cells exposed toT4 and control, according to the 3D Wound Healing Assay describedherein.

FIG. 10: depicts T4 increasing wound healing (measured by out migratingcells) in a dose-dependent manner between concentrations of 0.104 and1.004. This same increase is not seen in concentrations of T4 between1.004 and 3.004.

FIGS. 11A and 11B: depict the effect of unlabeled T4 and T3 on^(I-125)-T4 binding to purified integrin. Unlabeled T4 (10⁴M to 10⁻¹¹M)or T3 (10⁴M to 10⁻⁸M) were added to purified αVβ3 integrin (2 m/sample)and allowed to incubate for 30 min. at room temperature. Two microcuriesof I-125 labeled T4 was added to each sample. The samples were incubatedfor 20 min. at room temperature, mixed with loading dye, and run on a 5%Native gel for 24 hrs. at 4° C. at 45 mÅ. Following electrophoresis, thegels were wrapped in plastic wrap and exposed to film. ^(I-125)-T4binding to purified αVβ3 is unaffected by unlabeled T4 in the range of10⁻¹¹M to 10⁻⁷M, but is competed out in a dose-dependent manner byunlabeled T4 at a concentration of 10⁻⁶M. Radiolabeled T4 binding to theintegrin is almost completely displaced by 10⁴M unlabeled T4. T3 is lesseffective at competing out T4 binding to αVβ3, reducing the signal by11%, 16%, and 28% at 10⁻⁶M, 10⁻⁵M, and 10⁴M T3, respectively.

FIG. 12A: depicts tetrac and an RGD containing peptide, but not an RGEcontaining peptide compete out T4 binding to purified αVβ3. The tetracaddition to purified αVβ3 reduces ¹⁻¹²⁵-labeled T4 binding to theintegrin in a dose dependent manner. 10⁻⁸M tetrac is ineffective atcompeting out T4 binding to the integrin. The association of T4 and αVβ3was reduced by 38% in the presence of 10⁻⁷M tetrac and by 90% with 10⁻⁵Mtetrac. Addition of an RGD peptide at 10⁻⁵M competes out T4 binding toαVβ3. Application of 10⁻⁵M and 10⁴M RGE peptide, as a control for theRGD peptide, was unable to diminish T4 binding to purified αVβ3.

FIG. 12B: depicts a graphical representation of the tetrac and RGD datafrom FIG. 12A. Data points are shown as the mean±S.D. for 3 independentexperiments.

FIG. 13A: depicts the effects of the monoclonal antibody LM609 on T4binding to αVβ3. LM609 was added to αVβ3 at the indicatedconcentrations. One μg of LM609 per sample reduces ¹⁻¹²⁵-labeled T4binding to the integrin by 52%. Maximal inhibition of T4 binding to theintegrin is reached when concentrations of LM609 are 2 μg per sample andis maintained with antibody concentrations as high as 8 μg. As a controlfor antibody specificity, 10 μg/sample COX-2 mAB and 10 μg/sample mouseIgG were added to αVβ3 prior to incubation with T4.

FIG. 13B: depicts a graphical representation of data from FIG. 13A. Datapoints are shown as the mean±S.D. for 3 independent experiments.

FIG. 14A: depicts the effect of RGD, RGE, tetrac, and the mAB LM609 onT4-induced MAPK activation. CV-1 cells (50-70% confluency) were treatedfor 30 min. with 10⁻⁷ M T4 (10⁻⁷ M total concentration, 10⁻¹⁰M freeconcentration). Selected samples were treated for 16 hours with theindicated concentrations of either an RGD containing peptide, an RGEcontaining peptide, tetrac, or LM609 prior to the addition of T4.Nuclear proteins were separated by SDS-PAGE and immunoblotted withanti-phospho-MAPK (pERK1/2) antibody. Nuclear accumulation of pERK1/2 isdiminished in samples treated with 10⁻⁶ M RGD peptide or higher, but notsignificantly altered in samples treated with 10⁻⁴ M RGE. pERK1/2accumulation is decreased 76% in CV1 cells treated with 10⁻⁶M tetrac,while 10⁻⁵M and higher concentrations of tetrac reduce nuclearaccumulation of pERK1/2 to levels similar to the untreated controlsamples. The monoclonal antibody to αVβ3 LM609 decrease accumulation ofactivated MAPK in the nucleus when it is applied to CV1 cultures aconcentration of 1 μg/ml.

FIG. 14B: depicts a graphical representation of the data for RGD, RGE,and tetrac shown in FIG. 14A. Data points represent the mean±S.D. for 3separate experiments.

FIG. 15A: depicts the effects of siRNA to αV and β3 on T4 induced MAPKactivation. CV1 cells were transfected with siRNA (100 nM finalconcentration) to αV, β3, or αV and β3 together. Two days aftertransfection, the cells were treated with 10⁻⁷M T4. A) RT-PCR wasperformed from RNA isolated from each transfection group to verify thespecificity and functionality of each siRNA.

FIG. 15B: depicts nuclear proteins from each transfection of FIG. 15Awhich were isolated and subjected to SDS-PAGE.

FIG. 16A: depicts the inhibitory effect of αVβ3 mAB (LM609) onT4-stimulated angiogenesis in the CAM Model. Samples were exposed toPBS, T4 (0.1 μM), or T4 plus 10 mg/ml LM609 for 3 days. Angiogenesisstimulated by T4 is substantially inhibited by the addition of the αVβ3monoclonal antibody LM609.

FIG. 16B: depicts the tabulation of the mean±SEM of new branches formedfrom existing blood vessels during the experimental period of FIG. 16A.Data was drawn from 3 separate experiments, each containing 9 samples ineach treatment group.

FIGS. 16C and 16D: depicts angiogenesis stimulated by T4 or FGF2 is alsoinhibited by the addition of the αVβ3 monoclonal antibody LM609 or XT199.

FIG. 17: depicts a preparation of commercially available polyvinylalcohol (or related co-polymers) esterified by treatment with the acidchloride of thyroid hormone analogs, namely the acid chloride form. Thehydrochloride salt is neutralized by the addition of triethylamine toafford triethylamine hydrochloride which can be washed away with waterupon precipitation of the thyroid hormone ester polymer form fordifferent analogs.

FIG. 18: depicts a polymer covalent conjugation using an anhydridelinkage that is derived from reaction of an acrylic acid co-polymer.Neutralization of the hydrochloric acid is accomplished by treatmentwith triethylamine and subsequent washing of the precipitatedpolyanhydride polymer with water removes the triethylamine hydrochloridebyproduct. This reaction will lead to the formation of Thyroid hormoneanalog acrylic acid co-polymer+triethylamine.

FIG. 19: depicts the process of entrapment in a polylactic acid polymer.Polylactic acid polyester polymers (PLA) undergo hydrolysis in vivo tothe lactic acid monomer and this has been exploited as a vehicle fordrug delivery systems in humans. Unlike the prior two covalent methodswhere the thyroid hormone analog is linked by a chemical bond to thepolymer, this would be a non-covalent method that would encapsulate thethyroid hormone analog into PLA polymer beads. This reaction will leadto the formation of thyroid hormone analog containing PLA beads inwater. Filter and washing will result in the formation of thyroidhormone analog containing PLA beads, which upon in vivo hydrolysis willlead to the generation of controlled levels of thyroid hormone pluslactic acid.

FIGS. 20A, 20B, 20C and 20D: depict substitutions required to achievevarious thyroid hormone analogs which can be conjugated to createpolymeric forms of thyroid hormone analogs of the invention.

FIG. 21: depicts the induction of oxygen-induced retinopathy in a mousemode and immunohistochemical staining of ROP mice indicating normalvessels in the eye at room air, capillary drop out due to decreased VEGFat 75% oxygen, and neovascularization due to increased VEGF and otherangiogenic factors when mice are brought back to room air.

FIG. 22: depicts data representing the mean total area ofneovascularization after the administration of tetrac at 10 mg/kg, IP ornano-tetrac at 1 mg/kg, IP on day 12 and 15. Tetrac and nano-tetracresulted in approximately 50% inhibition of neovascularization.

FIG. 23A: depicts the effects on mice pups after exposure to room airand 75% O₂ on vascularization area of murine retinas showing increasedneovascularization.

FIG. 23B: depicts the effects of injected tetrac nanoparticles andtetrac on vascularization area of murine retinas showing suppression ofneovascularization.

FIG. 24A: depicts photomicrographs of representative chickchorioallantoic membranes treated with tetrac and tetrac-nanoparticlesat 2 m/CAM.

FIG. 24B: depicts graphically the quantification of the angiogenesisindex of the micrographs described in FIG. 24A by quantification ofangiogenic branch points under each treatment compared with the PBScontrol.

FIG. 25: depicts the CAM model of angiogenesis and a tumor implant,illustrating the induction of angiogenesis by LNCaP cancer cellsimplanted in matrigel.

FIG. 26: depicts graphically the ability of tetrac and tetracnanoparticles to inhibit tumor-induced angiogenesis by a determinationof hemoglobin concentration.

FIG. 27: depicts graphically the effects of tetrac on the proliferationof a drug resistant breast cancer xenograft.

FIG. 28A: depicts the effects of tetrac and tetrac-PLGA nanoparticles onLNCaP tumor growth in male mice xenografts.

FIG. 28B: depicts the effects of tetrac and tetrac-PLGA nanoparticles onLNCaP tumor angiogenesis in male mice xenografts.

FIG. 29: depicts comparison of tetrac administered at 1 mg/kg daily for17 days and paclitaxel administration for 17 days. Comparison showsdistinct suppression of prostate tumor growth by tetrac comparable tothe effects of the cytotoxic chemotherapeutic agent, paclitaxel.

FIG. 30: depicts the effects of tetrac and tetrac-nanoparticles on thecell proliferation of pancreatic cancer, Panc-1 cells. Cells werecultured for 8 days in the presence of vehicle (control), tetrac, ornanoparticulate tetrac (“NT”, wherein NT-1 and NT-2 denote separatelyprepared batches). Cells were counted on days 3, 6, and 8. By day 8,significant suppression of cell growth was observed in thetetrac-treated (*P<0.04) and NT-treated (**P<0.01) samples, compared tountreated controls.

FIG. 31A: depicts the up-regulation of pro-apoptotic bcl-X's proteinexpression by tetrac and tetrac-nanoparticle administration. Total cellextracts from treated Panc-1 cells were analyzed by immunoblotting forbcl-Xs protein. Significant accumulation of bcl-Xs protein was observedwith either unmodified tetrac or NT (*P<0.05).

FIG. 31B: depicts the up-regulation of anti-angiogenic THBS1 proteinexpression by tetrac and tetrac-nanoparticle administration. Cells weretreated for 3 days with control solvent, tetrac, or NT. Total celllysates were probed for THBS1. Both tetrac (P<0.05) and NT (P<0.01)increased the levels of THBS1.

FIG. 31C: depicts the up-regulation of cell cycle gene expression toslow proliferation by the administration of tetrac andtetrac-nanoparticles. Total RNA was used as the template for RT-PCR.Treatment of cells with either tetrac or nanoparticulate tetracdecreased p21 and p53 mRNA expression (p<0.05) and nanoparticulatetetrac alone significantly reduced EGFR expression levels.

FIG. 32A: depicts graphically, the anti-tumorigenic effects of tetracand tetrac-nanoparticles on pancreatic xenografts of AsPc-1 cells bycomparing the tumor volumes over a 15 day treatment period.

FIG. 32B: depicts a comparison of IVIS imaging of tumor masses of FIG.32A on day 15 after the final measurement was taken.

FIG. 32C: depicts the hemoglobin content as an index of tumorangiogenesis for the AsPc-1 cells xenografts depicted in FIG. 32A. Thehemoglobin content decreased by 60% in animals treated for 15 days witheither tetrac or tetrac-nanoparticles.

FIG. 33: depicts graphically the percentage of cell death of AsPc-1cells resistant to Gemcitabine, comparing untreated, Gemcitabine,tetrac-nanoparticles, and tetrac-nanoparticles+Gemcitabine combinationtreatment.

FIG. 34: depicts graphically the effects of tetrac and tetracnanoparticles on primary and metastatic non-small cell lung cancer tumorvolume.

FIG. 35A: depicts graphically the effects of tetrac and tetracnanoparticles on primary and metastatic non-small cell lung cancer tumorweight.

FIG. 35B: depicts graphically the effects of tetrac and tetracnanoparticles on primary and metastatic non-small cell lung cancer tumorhemoglobin content.

FIG. 36A: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor volume of 10×10⁶ primary and metastatic lungcancer cells.

FIG. 36B: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor weight of 10×10⁶ primary and metastatic lungcancer cells.

FIG. 37A: depicts graphically the effects of tetrac and tetracnanoparticles at 1 μg on the weight of primary and metastatic follicularthyroid carcinoma (FTC) tumors in the CAM model.

FIG. 37B: depicts graphically the effects of tetrac and tetracnanoparticles 1 μg on hemoglobin content of primary and metastaticfollicular thyroid carcinoma (FTC) tumors in the CAM model.

FIG. 37C: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor weight of primary and metastatic follicularthyroid carcinoma (FTC) tumors.

FIG. 38A: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor volume of primary and metastatic humanfollicular tumors growth in nude mice xenografts.

FIG. 38B: depicts graphically the tumor regrowth after discontinuing thetetrac and tetrac nanoparticles in FIG. 38A.

FIG. 39A: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor volume of primary and metastatic renal tumorsin nude mice xenografts.

FIG. 39B: depicts graphically tumor regrowth after discontinuingtreatment with tetrac and tetrac nanoparticles as depicted in FIG. 39A.

FIG. 40A: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor volume of renal cell carcinoma primary andmetastatic tumors.

FIG. 40B: depicts graphically the effects of tetrac and tetracnanoparticles on the tumor weight of renal cell carcinoma primary andmetastatic tumors.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described with references to the accompanying drawings, andas pointed out by the claims. For convenience, certain terms used in thespecification, examples and claims are collected here. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention pertains.

As used herein, the term “angiogenic agent” includes any compound orsubstance that promotes or encourages angiogenesis, whether alone or incombination with another substance. Examples include, but are notlimited to, T3, T4, T3-agarose or T4-agarose, polymeric analogs of T3,T4, 3,5-dimethyl-4-(4′-hydroy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or DITPA. In contrast, the terms “anti-angiogenesis agent” or“anti-angiogenic agent” refer to any compound or substance that inhibitsor discourages angiogenesis, whether alone or in combination withanother substance. Examples include, but are not limited to, tetrac,triac, XT 199, and mAb LM609.

As used herein, the term “myocardial ischemia” is defined as aninsufficient blood supply to the heart muscle caused by a decreasedcapacity of the heart vessels. As used herein, the term “coronarydisease” is defined as diseases/disorders of cardiac function due to animbalance between myocardial function and the capacity of coronaryvessels to supply sufficient blood flow for normal function. Specificcoronary diseases/disorders associated with coronary disease which canbe treated with the compositions and methods described herein includemyocardial ischemia, angina pectoris, coronary aneurysm, coronarythrombosis, coronary vasospasm, coronary artery disease, coronary heartdisease, coronary occlusion and coronary stenosis.

As used herein the term “occlusive peripheral vascular disease” (alsoknown as peripheral arterial occlusive disorder) is a vasculardisorder-involving blockage in the carotid or femoral arteries,including the iliac artery. Blockage in the femoral arteries causes painand restricted movement. A specific disorder associated with occlusiveperipheral vascular disease is diabetic foot, which affects diabeticpatients, often resulting in amputation of the foot.

As used herein the terms “regeneration of blood vessels,”“angiogenesis,” “revascularization,” and “increased collateralcirculation” (or words to that effect) are considered as synonymous. Theterm “pharmaceutically acceptable” when referring to a natural orsynthetic substance means that the substance has an acceptable toxiceffect in view of its much greater beneficial effect, while the relatedthe term, “physiologically acceptable,” means the substance hasrelatively low toxicity. The term, “co-administered” means two or moredrugs are given to a patient at approximately the same time or in closesequence so that their effects run approximately concurrently orsubstantially overlap. This term includes sequential as well assimultaneous drug administration.

“Pharmaceutically acceptable salts” refers to pharmaceuticallyacceptable salts of thyroid hormone analogs, polymeric forms, andderivatives, which salts are derived from a variety of organic andinorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetra-alkyl ammonium, and the like; and when the molecule contains abasic functionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like can be used as the pharmaceutically acceptablesalt.

“Subject” includes living organisms such as humans, monkeys, cows,sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, culturedcells therefrom, and transgenic species thereof. In a preferredembodiment, the subject is a human. Administration of the compositionsof the present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective totreat the condition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the age, sex, and weight of the subject,and the ability of the therapeutic compound to treat the foreign agentsin the subject. Dosage regimens can be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

“Administering” includes routes of administration which allow thecompositions of the invention to perform their intended function, e.g.,promoting angiogenesis. A variety of routes of administration arepossible including, but not necessarily limited to parenteral (e.g.,intravenous, intra-arterial, intramuscular, subcutaneous injection),oral (e.g., dietary), topical, nasal, rectal, intratumoral (directinjection into the tumor site), intraocular, or via slow releasingmicrocarriers depending on the disease or condition to be treated. Oral,parenteral and intravenous administration are preferred modes ofadministration. Formulation of the compound to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, gels, aerosols, capsule). An appropriate compositioncomprising the compound to be administered can be prepared in aphysiologically acceptable vehicle or carrier and optional adjuvants andpreservatives. For solutions or emulsions, suitable carriers include,for example, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media, sterile water, creams,ointments, lotions, oils, pastes, and solid carriers. Parenteralvehicles can include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles can include various additives, preservatives, orfluid, nutrient or electrolyte replenishers (See generally, Remington'sPharmaceutical Science, 16th Edition, Mack, Ed. (1980)).

“Effective amount” includes those amounts of pro-angiogenic oranti-angiogenic compounds which allow it to perform its intendedfunction, e.g., promoting or inhibiting angiogenesis inangiogenesis-related disorders as described herein. The effective amountwill depend upon a number of factors, including biological activity,age, body weight, sex, general health, severity of the condition to betreated, as well as appropriate pharmacokinetic properties. For example,dosages of the active substance may be from about 0.01 mg/kg/day toabout 500 mg/kg/day, advantageously from about 0.1 mg/kg/day to about100 mg/kg/day. A therapeutically effective amount of the activesubstance can be administered by an appropriate route in a single doseor multiple doses. Further, the dosages of the active substance can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation.

“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like which arecompatible with the activity of the compound and are physiologicallyacceptable to the subject. An example of a pharmaceutically acceptablecarrier is buffered normal saline (0.15M NaCl). The use of such mediaand agents for pharmaceutically active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the therapeutic compound, use thereof in the compositions suitablefor pharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

“Additional ingredients” include, but are not limited to, one or more ofthe following: excipients; surface active agents; dispersing agents;inert diluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed.

Compositions

Disclosed herein are angiogenic agents comprising thyroid hormones,analogs thereof, and polymer conjugations of the hormones and theiranalogs. The disclosed compositions can be used for promotingangiogenesis to treat disorders wherein angiogenesis is beneficial.Additionally, the inhibition of these thyroid hormones, analogs andpolymer conjugations can be used to inhibit angiogenesis to treatdisorders associated with such undesired angiogenesis. As used herein,the term “angiogenic agent” includes any compound or substance thatpromotes or encourages angiogenesis, whether alone or in combinationwith another substance. Examples include, but are not limited to, T3,T4, T3-agarose or T4-agarose, polymeric analogs of T3, T4,3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxyl acetic acid(GC-1), or DITPA.

Polymer conjugations are used to improve drug viability. While old andnew therapeutics are well-tolerated, many compounds need advanced drugdiscovery technologies to decrease toxicity, increase circulatory time,or modify biodistribution. One strategy for improving drug viability isthe utilization of water-soluble polymers. Various water-solublepolymers have been shown to modify biodistribution, improve the mode ofcellular uptake, change the permeability through physiological barriers,and modify the rate of clearance through the body. To achieve either atargeting or sustained-release effect, water-soluble polymers have beensynthesized that contain drug moieties as terminal groups, as part ofthe backbone, or as pendent groups on the polymer chain.

Representative compositions of the present invention include thyroidhormone or analogs thereof conjugated to polymers. Conjugation withpolymers can be either through covalent or non-covalent linkages. Inpreferred embodiments, the polymer conjugation can occur through anester, ether, anhydride, or sulfhydryl linkage. Representativecompositions of the present invention include thyroid hormone or analogsthereof conjugated to polymers. Conjugation with polymers can be eitherthrough covalent (i.e., ester, ether, sulfhydryl, or anhydride linkages)or non-covalent linkages. (See WO2008/140507, incorporated herein byreference, for specific examples).

An example of a polymer conjugation through an ester linkage usingpolyvinyl alcohol is shown in FIG. 17. In this preparation commerciallyavailable polyvinyl alcohol (or related co-polymers) can be esterifiedby treatment with the acid chloride of thyroid hormone analogs,including the acid chloride form. The hydrochloride salt is neutralizedby the addition of triethylamine to afford triethylamine hydrochloridewhich can be washed away with water upon precipitation of the thyroidhormone ester polymer form for different analogs. The ester linkage maystabilize the conjugated thyroid hormone analog nanoparticle and avoidlocal or systemic release of free thyroid hormone. Local release ofunmodified thyroid hormone may be undesired because of its intracellulareffects on mitochondrial energetics.

An example of a polymer conjugation through an anhydride linkage usingacrylic acid ethylene co-polymer is shown in FIG. 18. This is similar tothe previous polymer covalent conjugation, however, this time it isthrough an anhydride linkage that is derived from reaction of an acrylicacid co-polymer. Neutralization of the hydrochloric acid is accomplishedby treatment with triethylamine and subsequent washing of theprecipitated polyanhydride polymer with water removes the triethylaminehydrochloride byproduct. This reaction will lead to the formation ofthyroid hormone analog acrylic acid co-polymer+triethylamine. Similar tothe ester linkage, the anhydride linkage may also stabilize theconjugated thyroid hormones and avoid local or systemic release of freethyroid hormone. Instead the conjugated thyroid hormone will act at thecell surface receptor αVβ3 and it will not produce the intracellulareffects observed by unmodified thyroid hormones and analogs.

Another representative polymer conjugation includes thyroid hormone orits analogs conjugated to polyethylene glycol (PEG). Attachment of PEGto various drugs, proteins and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chains and viaother chemical methods. Peg itself, however, is limited to two activeagents per molecule. In a different approach, copolymers of PEG andamino acids were explored as novel biomaterials which would retain thebiocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule and which could besynthetically designed to suit a variety of applications.

Another representative polymer conjugation includes thyroid hormone orits analogs in non-covalent conjugation with polymers. This is shown indetail in FIG. 19. A preferred non-covalent conjugation is entrapment ofthyroid hormone or analogs thereof in a polylactic acid polymer.Polylactic acid polyester polymers (PLA) undergo hydrolysis in vivo tothe lactic acid monomer and this has been exploited as a vehicle fordrug delivery systems in humans. Unlike the prior two covalent methodswhere the thyroid hormone analog is linked by a chemical bond to thepolymer, this would be a non-covalent method that would encapsulate thethyroid hormone analog into PLA polymer beads. This reaction will leadto the formation of thyroid hormone analog containing PLA beads inwater. Filtering and washing will result in the formation of thyroidhormone analog containing PLA beads, which upon in vivo hydrolysis willlead to the generation of controlled levels of thyroid hormone pluslactic acid.

Furthermore, nanotechnology can be used for the creation of usefulmaterials and structures sized at the nanometer scale. The main drawbackwith biologically active substances is fragility. Nanoscale materialscan be combined with such biologically active substances to dramaticallyimprove the durability of the substance, create localized highconcentrations of the substance, and reduce costs by minimizing losses.Therefore, additional polymeric conjugations include nano-particleformulations of thyroid hormones and analogs thereof. In such anembodiment, nano-polymers and nano-particles can be used as a matrix forlocal delivery of thyroid hormone and its analogs. This will aid in timecontrolled delivery into the cellular and tissue target.

Compositions of the present invention include thyroid hormone, analogs,and derivatives either alone or in covalent or non-covalent conjugationwith polymers. Examples of representative analogs and derivatives areshown in FIG. 20, Tables A-D. Table A shows T2, T3, T4, andbromo-derivatives. Table B shows alanyl side chain modifications. TableC shows hydroxy groups, diphenyl ester linkages, and D-configurations.Table D shows tyrosine analogs.

The terms “anti-angiogenesis agent” or “anti-angiogenic agent” refer toany compound or substance that inhibits or discourages angiogenesis,whether alone or in combination with another substance. Examplesinclude, but are not limited to, tetrac, triac, XT 199, and mAb LM609.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inPromoting Angiogenesis

The pro-angiogenic effect of thyroid hormone analogs or polymeric formsdepends upon a non-genomic initiation, as tested by the susceptibilityof the hormonal effect to reduction by pharmacological inhibitors of theMAPK signal transduction pathway. Such results indicate that anotherconsequence of activation of MAPK by thyroid hormone is new blood vesselgrowth. The latter is initiated non-genomically, but of course, requiresa consequent complex gene transcription program. The ambientconcentrations of thyroid hormone are relatively stable. The CAM model,at the time we tested it, was thyroprival and thus may be regarded as asystem, which does not reproduce the intact organism.

The availability of a chick chorioallantoic membrane (CAM) assay forangiogenesis has provided a model in which to quantitate angiogenesisand to study possible mechanisms involved in the induction by thyroidhormone of new blood vessel growth. The present application discloses apro-angiogenic effect of T4 that approximates that in the CAM model ofFGF2 and that can enhance the action of suboptimal doses of FGF2. It isfurther disclosed that the pro-angiogenic effect of the hormone isinitiated at the plasma membrane and is dependent upon activation by T4of the MAPK signal transduction pathway. As provided above, methods fortreatment of occlusive peripheral vascular disease and coronarydiseases, in particular, the occlusion of coronary vessels, anddisorders associated with the occlusion of the peripheral vasculatureand/or coronary blood vessels are disclosed. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of thyroid hormone analogs, polymeric forms,and derivatives. The methods involve the co-administration of aneffective amount of thyroid hormone analogs, polymeric forms, andderivatives in low, daily dosages for a week or more with other standardpro-angiogenesis growth factors, vasodilators, anticoagulants,thrombolytics, or other vascular-related therapies.

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and compounds believed to promote angiogenesis. Forexample, T4 in physiological concentrations was shown to bepro-angiogenic in this in vitro model and on a molar basis to have theactivity of FGF2. The presence of 6-N-propyl-2-thiouracil (PTU) did notreduce the effect of T4, indicating that de-iodination of T4 to generateT3 was not a prerequisite in this model. A summary of thepro-angiogenesis effects of various thyroid hormone analogs is listed inTable 1.

TABLE 1 Pro-angiogenesis Effects of Various Thyroid Hormone Analogs inthe CAM Model TREATMENT ANGIOGENESIS INDEX PBS (Control) 89.4 ± 9.3DITPA (0.01 uM) 133.0 ± 11.6 DITPA (0.1 uM) 167.3 ± 12.7 DITPA (0.2 mM)117.9 ± 5.6  GC-1 (0.01 uM) 169.6 ± 11.6 GC-1 (0.1 uM) 152.7 ± 9.0  T4agarose (0.1 uM) 195.5 + 8.5  T4 (0.1 uM) 143.8 ± 7.9  FGF2 (1 ug) 155 ±9  n = 8 per group

The appearance of new blood vessel growth in this model requires severaldays, indicating that the effect of thyroid hormone was wholly dependentupon the interaction of the nuclear receptor for thyroid hormone (TR)with the hormone. Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T3, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T4 rather than T3, thenatural ligand of TR-raised the possibility that angiogenesis might beinitiated nongenomically at the plasma membrane by T4 and culminate ineffects that require gene transcription. Non-genomic actions of T4 havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand of iodothyronine and TR, but may interface with ormodulate gene transcription. Non-genomic actions of steroids have alsobeen well described and are known to interface with genomic actions ofsteroids or of other compounds. Experiments carried out with T4 andtetrac or with agarose-T4 indicated that the pro-angiogenic effect of T4indeed very likely was initiated at the plasma membrane. Tetrac blocksmembrane-initiated effects of T4, but does not, itself activate signaltransduction. Thus, it is a probe for non-genomic actions of thyroidhormone. Agarose-T4 has a molecular weight of 120,000 daltons and doesnot to gain entry to the cell interior and has been used to examinemodels for cell surface-initiated actions of the hormone. The thyroidhormone may be covalently bonded via the amino nitrogen on the alanineside chain of the agarose.

In part, this invention provides compositions and methods for promotingangiogenesis in a subject in need thereof. Conditions amenable totreatment by promoting angiogenesis include, for example, occlusiveperipheral vascular disease and coronary diseases, in particular, theocclusion of coronary vessels, and disorders associated with theocclusion of the peripheral vasculature and/or coronary blood vessels,erectile dysfunction, stroke, and wounds. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of polymeric forms of thyroid hormoneanalogs and derivatives and an effective amount of an adenosine and/ornitric oxide donor. The compositions can be in the form of a sterile,injectable, pharmaceutical formulation that includes an angiogenicallyeffective amount of thyroid hormone-like substance and adenosinederivatives in a physiologically and pharmaceutically acceptablecarrier, optionally with one or more excipients.

Myocardial Infarction

A major reason for heart failure following acute myocardial infarctionis an inadequate response of new blood vessel formation, i.e.,angiogenesis. Thyroid hormone and its analogs are beneficial in heartfailure and stimulate coronary angiogenesis. The methods of theinvention include, in part, delivering a single treatment of a thyroidhormone analog at the time of infarction either by direct injection intothe myocardium, or by simulation of coronary injection by intermittentaortic ligation to produce transient isovolumic contractions to achieveangiogenesis and/or ventricular remodeling.

Accordingly, in one aspect the invention features methods for treatingocclusive vascular disease, coronary disease, myocardial infarction,ischemia, stroke, and/or peripheral artery vascular disorders bypromoting angiogenesis by administering to a subject in need thereof anamount of a polymeric form of thyroid hormone, or an analog thereof,effective for promoting angiogenesis.

Examples of polymeric forms of thyroid hormone analogs are also providedherein and can include triiodothyronine (T3), levothyroxine (T4),(GC-1), or 3,5-diiodothyropropionic acid (DITPA) conjugated to polyvinylalcohol, acrylic acid ethylene co-polymer, polylactic acid, or agarose.

The methods also involve the co-administration of an effective amount ofthyroid hormone-like substance and an effective amount of an adenosineand/or NO donor in low, daily dosages for a week or more. One or bothcomponents can be delivered locally via catheter. Thyroid hormoneanalogs and derivatives in vivo can be delivered to capillary bedssurrounding ischemic tissue by incorporation of the compounds into anappropriately sized liposome, microparticle or nanoparticle. Thyroidhormone analogs, polymeric forms and derivatives can be targeted toischemic tissue by covalent linkage with a suitable antibody.

The method may be used as a treatment to restore cardiac function aftera myocardial infarction. The method may also be used to improve bloodflow in patients with coronary artery disease suffering from myocardialischemia or inadequate blood flow to areas other than the heartincluding, for example, occlusive peripheral vascular disease (alsoknown as peripheral arterial occlusive disease), or erectiledysfunction.

Wound Healing

Wound angiogenesis is an important part of the proliferative phase ofhealing. Healing of any skin wound other than the most superficialcannot occur without angiogenesis. Not only does any damaged vasculatureneed to be repaired, but the increased local cell activity necessary forhealing requires an increased supply of nutrients from the bloodstream.Moreover, the endothelial cells which form the lining of the bloodvessels are important in themselves as organizers and regulators ofhealing.

Thus, angiogenesis provides a new microcirculation to support thehealing wound. The new blood vessels become clinically visible withinthe wound space by four days after injury. Vascular endothelial cells,fibroblasts, and smooth muscle cells all proliferate in coordination tosupport wound granulation. Simultaneously, re-epithelialization occursto reestablish the epithelial cover. Epithelial cells from the woundmargin or from deep hair follicles migrate across the wound andestablish themselves over the granulation tissue and provisional matrix.Growth factors such as keratinocyte growth factor (KGF) mediate thisprocess. Several models (sliding versus rolling cells) ofepithelialization exist.

As thyroid hormones regulate metabolic rate, when the metabolism slowsdown due to hypothyroidism, wound healing also slows down. The role oftopically applied thyroid hormone analogs or polymeric forms in woundhealing therefore represents a novel strategy to accelerate woundhealing in diabetics and in non-diabetics with impaired wound healingabilities. Topical administration can be in the form of attachment to aBand-Aid. Additionally, nano-polymers and nano-particles prevent thethyroid hormone and analogs thereof from entering the intracellularportions of the cell thus reducing the side effects exhibited throughgenomic actions on gene expression.

Accordingly, another embodiment of the invention features methods fortreating wounds by promoting angiogenesis by administering to a subjectin need thereof an amount of a polymeric form of thyroid hormone, or ananalog thereof, effective for promoting angiogenesis. For details, seeExample 9.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inInhibiting Angiogenesis

The invention also provides, in another part, compositions and methodsfor inhibiting angiogenesis in a subject in need thereof. Conditionsamenable to treatment by inhibiting angiogenesis include, for example,primary or metastatic tumors and diabetic retinopathy. The compositionscan include an effective amount of tetrac, triac, or mAb LM609. Thecompositions can be in the form of a sterile, injectable, pharmaceuticalformulation that includes an anti-angiogenically effective amount of ananti-angiogenic substance in a physiologically and pharmaceuticallyacceptable carrier, optionally with one or more excipients. In a furtheraspect, the invention provides methods for treating a condition amenableto treatment by inhibiting angiogenesis by administering to a subject inneed thereof an amount of an anti-angiogenesis agent effective forinhibiting angiogenesis.

Examples of the anti-angiogenesis agents used for inhibitingangiogenesis are also provided by the invention and include, but are notlimited to, tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid(triac), monoclonal antibody LM609, or combinations thereof. Suchanti-angiogenesis agents can act at the cell surface to inhibit thepro-angiogenesis agents.

Cancer-Related New Blood Vessel Growth

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to, primary or metastatictumors. In such a method, compounds which inhibit the thyroidhormone-induced angiogenic effect are used to inhibit angiogenesis.Details of such a method are illustrated in Examples 12, 14, 16, 17, 20,and 21.

Diabetic Retinopathy

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to diabetic retinopathy, andrelated conditions. In such a method, compounds which inhibit thethyroid hormone-induced angiogenic effect are used to inhibitangiogenesis. Details of such a method and experimental data confirmingtreatment of diabetic retinopathy are illustrated in Examples 8A, 8B and13. While the provided examples are illustrated using tetrac,nanoparticulate tetrac or polymers thereof conjugated via a covalentbond, triac and mAb LM609 and nanoparticle formulations and polymerformulations thereof may be substituted in place of tetrac and achievesimilar results.

Retinopathy of Prematurity (ROP) is a blindness-causing neovascularizingdisease affecting premature infants treated with high concentrations ofoxygen. ROP develops in two distinct stages: 1) the hyperoxic insultleads to obliteration of immature retinal vessels and 2) initiated uponresumption of the breathing of normal air, is an adverse compensatoryneovascularization response. The formation of new vessels is excessive,neovessels may be leaky, and the inner limiting membrane of the retinamay be breached, allowing vessel growth into the vitreous which mightultimately lead to retinal detachment and vision loss. The formation ofnew vessels is mediated by ischemia-induced vascular endothelial growthfactor (VEGF). VEGF is a potent and specific endothelial cell cytokinethat can be up-regulated by hypoxia. Evidences show that VEGF is asignificant mediator in retinal neovascular diseases and other disordersin which hypoxia is believed to influence the pathogenesis. Tetrac is apotent inhibitor of VEGF or other growth factor-induced angiogenesis.

It is known that proliferative retinopathy induced by hypoxia (ratherthan diabetes) depends upon alphaV (αV) integrin expression. Thyroidhormone action on a specific integrin alpha-V-beta-3 (αVβ3) ispermissive in the development of diabetic retinopathy. Integrin αVβ3contains the cell surface receptor for thyroid hormone and thyroidhormone analogues. Thyroid hormone, its analogues, and polymerconjugations, act via this receptor to induce angiogenesis.

Methods of Treatment

Thyroid hormone analogs, polymeric forms, and derivatives can be used ina method for promoting angiogenesis in a patient in need thereof. Themethod involves the co-administration of an effective amount of thyroidhormone analogs, polymeric forms, and derivatives in low, daily dosagesfor a week or more. The method may be used as a treatment to restorecardiac function after a myocardial infarction. The method may also beused to improve blood flow in patients with coronary artery diseasesuffering from myocardial ischemia or inadequate blood flow to areasother than the heart, for example, peripheral vascular disease, forexample, peripheral arterial occlusive disease, where decreased bloodflow is a problem.

The compounds can be administered via any medically acceptable meanswhich is suitable for the compound to be administered, including oral,rectal, topical, intraocular, intranasal, intratumoral or parenteral(including subcutaneous, intramuscular and intravenous) administration.For example, adenosine has a very short half-life. For this reason, itis preferably administered intravenously. However, adenosine agonistshave been developed which have much longer half-lives, and which can beadministered through other means. Thyroid hormone analogs, polymericforms, and derivatives can be administered, for example, intravenously,orally, topically, intraocularly, intratumorally or intranasally.

In some embodiments, the thyroid hormone analogs, polymeric forms, andderivatives are administered via different means.

The amounts of the thyroid hormone, its analogs, polymeric forms, andderivatives required to be effective in stimulating angiogenesis will,of course, vary with the individual being treated and is ultimately atthe discretion of the physician. The factors to be considered includethe condition of the patient being treated, the efficacy of theparticular adenosine A₂ receptor agonist being used, the nature of theformulation, and the patient's body weight. Occlusion-treating dosagesof thyroid hormone analogs or its polymeric forms, and derivatives areany dosages that provide the desired effect.

Formulations

The compounds described above are preferably administered in aformulation including thyroid hormone analogs or its polymeric forms,and derivatives together with an acceptable carrier for the mode ofadministration. Any formulation or drug delivery system containing theactive ingredients, which is suitable for the intended use, as aregenerally known to those of skill in the art, can be used. Suitablepharmaceutically acceptable carriers for oral, rectal, topical,parenteral (including subcutaneous, intraperitoneal, intramuscular andintravenous), or intratumoral (direct injection to the tumor site)administration are known to those of skill in the art. The carrier mustbe pharmaceutically acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

Formulations suitable for parenteral administration and intratumoraladministration conveniently include sterile aqueous preparation of theactive compound, which is preferably isotonic with the blood of therecipient. Thus, such formulations may conveniently contain distilledwater, 5% dextrose in distilled water or saline. Useful formulationsalso include concentrated solutions or solids containing the compoundwhich upon dilution with an appropriate solvent give a solution suitablefor parental administration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion, ora draught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface active,or dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the thyroid hormone analogs or its polymeric forms,and adenosine derivatives can be formulated into a liposome,microparticle or nanoparticle, which is suitably sized to lodge incapillary beds following intravenous administration. When the liposome,microparticle or nanoparticle is lodged in the capillary bedssurrounding ischemic tissue, the agents can be administered locally tothe site at which they can be most effective. Suitable liposomes fortargeting ischemic tissue are generally less than about 200 nanometersand are also typically unilamellar vesicles, as disclosed, for example,in U.S. Pat. No. 5,593,688 to Baldeschweiler, entitled “Liposomaltargeting of ischemic tissue,” the contents of which are herebyincorporated by reference.

Preferred microparticles are those prepared from biodegradable polymers,such as polyglycolide, polylactide and copolymers thereof. Those ofskill in the art can readily determine an appropriate carrier systemdepending on various factors, including the desired rate of drug releaseand the desired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half-life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered directly to the inside of the tissue lumen.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations can optionally include additional components, such asvarious biologically active substances such as growth factors (includingTGF-beta., basic fibroblast growth factor (FGF2), epithelial growthfactor (EGF), transforming growth factors alpha and beta (TGF alpha. andbeta.), nerve growth factor (NGF), platelet-derived growth factor(PDGF), and vascular endothelial growth factor/vascular permeabilityfactor (VEGF/VPF)), antiviral, antibacterial, anti-inflammatory,immuno-suppressant, analgesic, vascularizing agent, and cell adhesionmolecule.

In addition to the aforementioned ingredients, the formulations mayfurther include one or more optional accessory ingredient(s) utilized inthe art of pharmaceutical formulations, e.g., diluents, buffers,flavoring agents, binders, surface active agents, thickeners,lubricants, suspending agents, preservatives (including antioxidants),and the like.

Materials & Methods

Reagents:

All reagents were chemical grade and purchased from Sigma Chemical Co.(St. Louis, Mo.) or through VWR Scientific (Bridgeport, N.J.). Cortisoneacetate, bovine serum albumin (BSA) and gelatin solution (2% type B frombovine skin) were purchased from Sigma Chemical Co. Fertilized chickeneggs were purchased from Charles River Laboratories, SPAFAS AvianProducts & Services (North Franklin, Conn.). T4,3,5,3′-triiodo-L-thyronine (T3), tetraiodothyroacetic acid (tetrac),T4-agarose, and 6-N-propyl-2-thiouracil (PTU) were obtained from Sigma;PD 98059 from Calbiochem; and CGP41251 was a gift from Novartis Pharma(Basel, Switzerland). Polyclonal anti-FGF2 and monoclonal anti-β-actinwere obtained from Santa Cruz Biotechnology and human recombinant FGF2from Invitrogen. Polyclonal antibody to phosphorylated ERK1/2 was fromNew England Biolabs and goat anti-rabbit IgG from DAKO.

Chorioallantoic Membrane (CAM) Model of Angiogenesis:

In vivo Neovascularization was examined by methods described previously.9-12 Ten-day-old chick embryos were purchased from SPAFAS (Preston,Conn.) and incubated at 37° C. with 55% relative humidity. A hypodermicneedle was used to make a small hole in the shell concealing the airsac, and a second hole was made on the broad side of the egg, directlyover an avascular portion of the embryonic membrane that was identifiedby candling. A false air sac was created beneath the second hole by theapplication of negative pressure at the first hole, causing the CAM toseparate from the shell. A window approximately 1.0 cm² was cut in theshell over the dropped CAM with a small-crafts grinding wheel (Dremel,division of Emerson Electric Co.), allowing direct access to theunderlying CAM. FGF2 (1 μg/mL) was used as a standard proangiogenicagent to induce new blood vessel branches on the CAM of 10-day-oldembryos. Sterile disks of No. 1 filter paper (Whatman International)were pretreated with 3 mg/mL cortisone acetate and 1 mmol/L PTU and airdried under sterile conditions. Thyroid hormone, hormone analogues, FGF2or control solvents, and inhibitors were then applied to the disks andthe disks allowed to dry. The disks were then suspended in PBS andplaced on growing CAMs. Filters treated with T4 or FGF2 were placed onthe first day of the 3-day incubation, with antibody to FGF2 added 30minutes later to selected samples as indicated. At 24 hours, the MAPKcascade inhibitor PD 98059 was also added to CAMs topically by means ofthe filter disks.

Microscopic Analysis of CAM Sections:

After incubation at 37° C. with 55% relative humidity for 3 days, theCAM tissue directly beneath FGF2 and TF/VIIa filter disk was resectedfrom control and treated CAM samples. Tissues were washed 3× with PBS,placed in 35-mm Petri dishes (Nalge Nunc), and examined under an SV6stereomicroscope (Zeiss) at X50 magnification. Digital images of CAMsections exposed to filters were collected using a 3-charge—coupleddevice color video camera system (Toshiba) and analyzed with Image-Prosoftware (Media Cybernetics). The number of vessel branch pointscontained in a circular region equal to the area of each filter diskwere counted. One image was counted in each CAM preparation, andfindings from 8 to 10 CAM preparations were analyzed for each treatmentcondition (thyroid hormone or analogues, FGF2, FGF2 antibody, PD 98059).In addition, each experiment was performed 3 times. The resultingangiogenesis index is the mean±SEM of new branch points in each set ofsamples.

Results:

Effects of topically applied formulations of tetrac andtetrac-nanoparticles on Fibroblast Growth Factor (FGF2) inhibitedangiogenesis in the CAM model. Tetrac or tetrac immobilized on PLGAnanoparticles were administered to chick embryos topically, onto growthfactor (FGF)-impregnated filter disk placed over the branch point of aselected vessel. After 3 days, CAM tissues directly beneath the growthfactor filter disk were removed, examined microscopically, and theimages analyzed with the Image-Pro Plus software. The number of branchpoints in a circular region equal to the area of a filter was counted.Nanoparticulate tetrac and tetrac significantly inhibited branch pointformation-induced by FGF2

FGF2 Assays:

ECV304 endothelial cells were cultured in M199 medium supplemented with10% fetal bovine serum. ECV304 cells (10⁶ cells) were plated on 0.2%gel-coated 24-well plates in complete medium overnight, and the cellswere then washed with serum-free medium and treated with T4 or T3 asindicated. After 72 hours, the supernatants were harvested and assaysfor FGF performed without dilution using a commercial ELISA system (R&DSystems).

MAPK Activation:

ECV304 endothelial cells were cultured in M199 medium with 0.25%hormone-depleted serum 13 for 2 days. Cells were then treated with T4(10⁻⁷ mol/L) for 15 minutes to 6 hours. In additional experiments, cellswere treated with T4 or FGF2 or with T4 in the presence of PD 98059 orCGP41251. Nuclear fractions were prepared from all samples by our methodreported previously, the proteins separated by polyacrylamide gelelectrophoresis, and transferred to membranes for immunoblotting withantibody to phosphorylated ERK 1/2. The appearance of nuclearphosphorylated ERK1/2 signifies activation of these MAPK isoforms by T4.

Reverse Transcription—Polymerase Chain Reaction:

Confluent ECV304 cells in 10-cm plates were treated with T4 (10⁻⁷ mol/L)for 6 to 48 hours and total RNA extracted using guanidiniumisothiocyanate (Biotecx Laboratories). RNA (1 μg) was subjected toreverse transcription-polymerase chain reaction (RT-PCR) using theAccess RT-PCR system (Promega). Total RNA was reverse transcribed intocDNA at 48° C. for 45 minutes, then denatured at 94° C. for 2 minutes.Second-strand synthesis and PCR amplification were performed for 40cycles with denaturation at 94° C. for 30 s, annealing at 60° C. for 60s, and extension at 68° C. for 120 s, with final ex-tension for 7minutes at 68° C. after completion of all cycles. PCR primers for FGF2were as follows: FGF2 sense strand 5′-TGGTATGTGGCACTGAAACG-3′ (SEQ IDNO:1), antisense strand 5′ CTCAATGACCTGGCGAAGAC-3′ (SEQ ID NO:2); thelength of the PCR product was 734 bp. Primers for GAPDH included thesense strand 5′-AAGGTCATCCCTGAGCTGAACG-3′ (SEQ ID NO:3), and antisensestrand 5′-GGGTGTCGCTGTTGAAGTCAGA-3′ (SEQ ID NO:4); the length of the PCRproduct was 218 bp. The products of RT-PCR were separated byelectrophoresis on 1.5% agarose gels and visualized with ethidiumbromide. The target bands of the gel were quantified using LabImagesoftware (Kapelan), and the value for [FGF2/GAPDH]X10 calculated foreach time point.

Statistical Analysis:

Statistical analysis was performed by 1-way ANOVA comparing experimentalwith control samples.

In Vivo Angiogenesis in Matrigel FGF₂ or Cancer Cell Lines Implant inMice: In Vivo Murine Angiogenesis Model:

The murine matrigel model will be conducted according to previouslydescribed methods (Grant et al., 1991; Okada et al., 1995) and asimplemented in our laboratory (Powel et al., 2000). Briefly, growthfactor free matrigel (Becton Dickinson, Bedford Mass.) will be thawedovernight at 4° C. and placed on ice. Aliquots of matrigel will beplaced into cold polypropylene tubes and FGF2, thyroid hormone analogsor cancer cells (1×10⁶ cells) will be added to the matrigel. Matrigelwith saline, FGF2, thyroid hormone analogs or cancer cells will besubcutaneously injected into the ventral midline of the mice. At day 14,the mice will be sacrificed and the solidified gels will be resected andanalyzed for presence of new vessels. T4, T4-agarose, FgF2 Control inPBS and thyroid inhibitor tetrac and nanoparticulate tetrac dissolved inPBS will be injected subcutaneously at different doses. Control andexperimental gel implants will be placed in a micro centrifuge tubecontaining 0.5 ml of cell lysis solution (Sigma, St. Louis, Mo.) andcrushed with a pestle. Subsequently, the tubes will be allowed toincubate overnight at 4° C. and centrifuged at 1,500×g for 15 minutes onthe following day. A 200 μl aliquot of cell lysate will be added to 1.3ml of Drabkin's reagent solution (Sigma, St. Louis, Mo.) for eachsample. The solution will be analyzed on a spectrophotometer at a 540nm. The absorption of light is proportional to the amount of hemoglobincontained in the sample.

Tumor Growth and Metastasis—Chick Chorioallantoic Membrane (CAM) Modelof Tumor Implant:

The protocol is as previously described (Kim et al., 2001). Briefly,1×10⁷ tumor cells will be placed on the surface of each CAM (7 day oldembryo) and incubated for one week. The resulting primary and metastatictumors will be excised and cut into 50 mg fragments. These fragments areplaced on additional 10 CAMs per group and treated topically thefollowing day with 25 μl of T4, T4-agarose, FGF2 Control in PBS andthyroid inhibitor tetrac and nanoparticulate tetrac dissolved in PBS.Seven days later, tumors will then be excised from the egg and tumorweights will be determined for each CAM. FIG. 8 is a diagrammatic sketchshowing the steps involved in the in vivo tumor growth model in the CAM.

The effects of tetrac, triac, and thyroid hormone antagonists on tumorgrowth rate, tumor angiogenesis, and tumor metastasis of cancer celllines can be determined.

Tumor Growth and Metastasis—Tumor Xenograft Model in Mice:

The model is as described in our publications by Kerr et al., 2000; VanWaes et al., 2000; Ali et al., 2001; and Ali et al., 2001, each of whichis incorporated herein by reference in its entirety). The anti-cancerefficacy for tetrac, triac, and other thyroid hormone antagonists atdifferent doses and against different primary and metastatic tumor typescan be determined and compared.

Tumor Growth and Metastasis—Experimental Model of Metastasis:

The model is as described in our recent publications (Mousa, 2002;Amirkhosravi et al., 2003a and 2003b, each of which is incorporated byreference herein in its entirety). Briefly, B16 murine malignantmelanoma cells (ATCC, Rockville, Md.) and other cancer lines will becultured in RPMI 1640 (Invitrogen, Carlsbad, Calif.), supplemented with10% fetal bovine serum, penicillin and streptomycin (Sigma, St. Louis,Mo.). Cells will be cultured to 70% confluency and harvested withtrypsin-EDTA (Sigma) and washed twice with phosphate buffered saline(PBS). Cells were re-suspended in PBS at a concentration of either2.0×10⁵ cells/ml for experimental metastasis. Animals: C57/BL6 mice(Harlan, Indianapolis, Ind.) weighing 18-21 grams were used for thisstudy. All procedures are in accordance with IACUC and institutionalguidelines. The anti-cancer efficacy for tetrac, triac, and otherthyroid hormone antagonists at different doses and against differenttumor types can be determined and compared.

Effect of Thyroid Hormone Analogues on Angiogenesis.

T4 induced significant increase in angiogenesis index (fold increaseabove basal) in the CAM model. T3 at 0.001-1.0 μM or T4 at 0.1-1.0 μMachieved maximal effect in producing 2-2.5 fold increase in angiogenesisindex as compared to 2-3 fold increase in angiogenesis index by 1 μg ofFGF2 (Table 1 and FIGS. 1 a and 1 b). The effect of T4 in promotingangiogenesis (2-2.5 fold increase in angiogenesis index) was achieved inthe presence or absence of PTU, which inhibit T4 to T3 conversion. T3itself at 91-100 nM-induced potent pro-angiogenic effect in the CAMmodel. T4-agarose produced similar pro-angiogenesis effect to thatachieved by T4. The pro-angiogenic effect of either T4 or T4-agarose was100% blocked by tetrac or triac.

Enhancement of Pro-Angiogenic Activity of FGF2 by Sub-MaximalConcentrations of T4.

The combination of T4 and FGF2 at sub-maximal concentrations resulted inan additive increase in the angiogenesis index up to the same level likethe maximal pro-angiogenesis effect of either FGF2 or T4 (FIG. 2).

Effects of MAPK Cascade Inhibitors on the Pro-Angiogenic Actions of T4and FGF2 in the CAM model.

The pro-angiogenesis effect of either T4 or FGF2 was totally blocked byPD 98059 at 0.8 (angiogenesis inhibitor)—8 μg (FIG. 3).

Effects of Specific Integrin αvβ3 Antagonists on the Pro-AngiogenicActions of T4 and FGF2 in the CAM Model.

The pro-angiogenesis effect of either T4 or FGF2 was totally blocked bythe specific monoclonal antibody LM609 at 10 μg (FIGS. 4 a and 4 b).

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and other promoters or inhibitors of angiogenesis. Inthe present studies, T4 in physiological concentrations was shown to bepro-angiogenic, with comparable activity to that of FGF2. The presenceof PTU did not reduce the effect of T4, indicating that de-iodination ofT4 to generate T3 was not a prerequisite in this model. Because theappearance of new blood vessel growth in this model requires severaldays, we assumed that the effect of thyroid hormone was totallydependent upon the interaction of the nuclear receptor for thyroidhormone (TR). Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T3, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T4—rather than T3, thenatural ligand of TR, raised the possibility that angiogenesis might beinitiated non-genomically at the plasma membrane by T4 and culminate ineffects that require gene transcription. Non-genomic actions of T4 havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand binding of iodothyronine and TR, but may interfacewith or modulate gene transcription. Non-genomic actions of steroidshave also been well-described and are known to interface with genomicactions of steroids or of other compounds. Experiments carried out withT4 and tetrac or with agarose-T4 indicated that the pro-angiogeniceffect of T4 indeed very likely was initiated at the plasma membrane. Wehave shown elsewhere that tetrac blocks membrane-initiated effects ofT4, but does not, itself, activate signal transduction. Thus, it is aprobe for non-genomic actions of thyroid hormone. Agarose-T4 is thoughtnot to gain entry to the cell interior and has been used by us andothers to examine models for possible cell surface-initiated actions ofthe hormone.

These results suggest that another consequence of activation of MAPK bythyroid hormone is new blood vessel growth. The latter is initiatednon-genomically, but of course requires a consequent complex genetranscription program.

The ambient concentrations of thyroid hormone are relatively stable. TheCAM model, at the time we tested it, was thyroprival and thus may beregarded as a system, which does not reproduce the intact organism. Wepropose that circulating levels of T4 serve, with a variety of otherregulators, to modulate the sensitivity of vessels to endogenousangiogenic factors, such as VEGF and FGF2.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLES Example 1 Effect of Thyroid Hormone on Angiogenesis

As seen in FIG. 1A and summarized in FIG. 1B, both L-T4 and L-T3enhanced angiogenesis in the CAM assay. T4, at a physiologic totalconcentration in the medium of 0.1 μM, increased blood vessel branchformation by 2.5-fold (P<0.001). T3 (1 nM) also stimulated angiogenesis2-fold. The possibility that T4 was only effective because of conversionof T4 to T3 by cellular 5′-monodeiodinase was ruled out by the findingthat the deiodinase inhibitor PTU had no inhibitory effect onangiogenesis produced by T4. PTU was applied to all filter disks used inthe CAM model. Thus, T4 and T3 promote new blood vessel branch formationin a CAM model that has been standardized previously for the assay ofgrowth factors.

Example 2 Effects of T4—Agarose and Tetrac

We have shown previously that T4-agarose stimulates cellular signaltransduction pathways initiated at the plasma membrane in the samemanner as T4 and that the actions of T4 and T4-agarose are blocked by adeaminated iodothyronine analogue, tetrac, which is known to inhibitbinding of T4 to plasma membranes. In the CAM model, the addition oftetrac (0.1 μM) inhibited the action of T4 (FIG. 2A), but tetrac alonehad no effect on angiogenesis (FIG. 2C). The action of T4-agarose, addedat a hormone concentration of 0.1 μM, was comparable to that of T4 inthe CAM model (FIG. 2B), and the effect of T4-agarose was also inhibitedby the action of tetrac (FIG. 2B; summarized in 2C).

Example 3 Enhancement of Proangiogenic Activity of FGF2 by a SubmaximalConcentration of T4

Angiogenesis is a complex process that usually requires theparticipation of polypeptide growth factors. The CAM assay requires atleast 48 hours for vessel growth to be manifest; thus, the apparentplasma membrane effects of thyroid hormone in this model are likely toresult in a complex transcriptional response to the hormone. Therefore,we determined whether FGF2 was involved in the hormone response andwhether the hormone might potentiate the effect of subphysiologic levelsof this growth factor. T4 (0.05 μM) and FGF2 (0.5 μg/mL) individuallystimulated angiogenesis to a modest degree (FIG. 3). The angiogeniceffect of this submaximal concentration of FGF2 was enhanced by asubphysiologic concentration of T4 to the level caused by 1.0 μg FGF2alone. Thus, the effects of submaximal hormone and growth factorconcentrations appear to be additive. To define more precisely the roleof FGF2 in thyroid hormone stimulation of angiogenesis, a polyclonalantibody to FGF2 was added to the filters treated with either FGF2 orT4, and angiogenesis was measured after 72 hours. FIG. 4 demonstratesthat the FGF2 antibody inhibited angiogenesis stimulated either by FGF2or by T4 in the absence of exogenous FGF2, suggesting that the T4 effectin the CAM assay was mediated by increased FGF2 expression. Control IgGantibody has no stimulatory or inhibitory effect in the CAM assay.

Example 4 Stimulation of FGF2 Release from Endothelial Cells by ThyroidHormone

Levels of FGF2 were measured in the media of ECV304 endothelial cellstreated with either T4 (0.1 μM) or T3 (0.01 μM) for 3 days. As seen inthe Table 2, T3 stimulated FGF2 concentration in the medium 3.6-fold,whereas T4 caused a 1.4-fold increase. This finding indicates thatthyroid hormone may enhance the angiogenic effect of FGF2, at least inpart, by increasing the concentration of growth factor available toendothelial cells.

TABLE 2 Effect of T4 and T3 on Release of FGF2 From ECV304 EndothelialCells Cell Treatment FGF2 (pg/mL/10⁶ cells) Control 27.7 ± 3.1 T3 (0.01μM)  98.8 ± 0.5* T3 + PD 98059 (2 μM) 28.4 ± 3.2 T3 + PD 98059 (20 μM)21.7 ± 3.5 T4 (0.1 μM)  39.2 ± 2.8† T4 + PD 98059 (2 μM) 26.5 ± 4.5 T4 +PD 98059 (20 μM) 23.2 ± 4.8 *P < 0.001, comparing T3-treated sampleswith control samples by ANOVA; †P < 0.05, comparing T4-treated sampleswith control samples by ANOVA.

Example 5 Role of the ERK1/2 Signal Transduction Pathway in Stimulationof Angiogenesis by Thyroid Hormone and FGF2

A pathway by which T4 exerts a non-genomic effect on cells is the MAPKsignal transduction cascade, specifically that of ERK1/2 activation. Weknow that T4 enhances ERK1/2 activation by epidermal growth factor. Therole of the MAPK pathway in stimulation by thyroid hormone of FGF2expression was examined by the use of PD 98059 (2 to 20 μM), aninhibitor of ERK1/2 activation by the tyrosine-threonine kinases MAPKkinase-1 (MEK1) and MEK2. The data in the Table demonstrate that PD98059 effectively blocked the increase in FGF2 release from ECV304endothelial cells treated with either T4 or T3. Parallel studies ofERK1/2 inhibition were performed in CAM assays, and representativeresults are shown in FIG. 5. A combination of T3 and T4, each inphysiologic concentrations, caused a 2.4-fold increase in blood vesselbranching, an effect that was completely blocked by 3 μM PD 98059 (FIG.5A). FGF2 stimulation of branch formation (2.2-fold) was alsoeffectively blocked by this inhibitor of ERK1/2 activation (FIG. 5B).Thus, the proangiogenic effect of thyroid hormone begins at the plasmamembrane and involves activation of the ERK1/2 pathway to promote FGF2release from endothelial cells. ERK1/2 activation is again required totransduce the FGF2 signal and cause new blood vessel formation.

Example 6 Action of Thyroid Hormone and FGF2 on MAPK Activation

Stimulation of phosphorylation and nuclear translocation of ERK1/2 MAPKswas studied in ECV304 cells treated with T4 (10⁻⁷ mol/L) for 15 minutesto 6 hours. The appearance of phosphorylated ERK1/2 in cell nucleioccurred within 15 minutes of T4 treatment, reached a maximal level at30 minutes, and was still apparent at 6 hours (FIG. 6A). This effect ofthe hormone was inhibited by PD 98059 (FIG. 6B), a result to be expectedbecause this compound blocks the phosphorylation of ERK1/2 by MAPKkinase. The traditional protein kinase C (PKC)-α, PKC-β, and PKC-γinhibitor CGP41251 also blocked the effect of the hormone on MAPKactivation in these cells, as we have seen with T4 in other cell lines.Thyroid hormone enhances the action of several cytokines and growthfactors, such as interferon-γ13 and epidermal growth factor. In ECV304cells, T4 enhanced the MAPK activation caused by FGF2 in a 15-minute coincubation (FIG. 6C). Applying observations made in ECV304 cells to theCAM model, we propose that the complex mechanism by which the hormoneinduces angiogenesis includes endothelial cell release of FGF2 andenhancement of the autocrine effect of released FGF2 on angiogenesis.

Example 7 RT-PCR in ECV304 Cells Treated with Thyroid Hormone

The final question addressed in studies of the mechanism of theproangiogenic action of T4 was whether the hormone may induce FGF2 geneexpression. Endothelial cells were treated with T4 (10⁻⁷ mol/L) for 6 to48 hours, and RT-PCR—based estimates of FGF2 and GAPDH RNA (inferredfrom cDNA measurements; FIG. 7) were performed. Increase in abundance ofFGF2 cDNA, corrected for GAPDH content, was apparent by 6 hours ofhormone treatment and was further enhanced by 48 hours.

Example 8A Retinal Neovascularization Model in Mice (Diabetic andNon-Diabetic)

To assess the pharmacologic activity of a test article on retinalneovascularization, infant mice are exposed to a high oxygen environmentfor 7 days and allowed to recover, thereby stimulating the formation ofnew vessels on the retina. Test articles are evaluated to determine ifretinal neovascularization is suppressed. The retinas are examined withhematoxylin-eosin staining and with at least one stain, whichdemonstrates neovascularization (usually a Selectin stain). Other stains(such as PCNA, PAS, GFAP, markers of angiogenesis, etc.) can be used. Asummary of the model is below:

Animal Model

-   -   Infant mice (P7) and their dams are placed in a hyper-oxygenated        environment (70-80%) for 7 days.    -   On P12, the mice are removed from the oxygenated environment and        placed into a normal environment    -   Mice are allowed to recover for 5-7 days.    -   Mice are then sacrificed and the eyes collected.    -   Eyes are either frozen or fixed as appropriate    -   The eyes are stained with appropriate histochemical stains    -   The eyes are stained with appropriate immunohistochemical stains    -   Blood, serum, or other tissues can be collected    -   Eyes, with special reference to microvascular alterations, are        examined for any and all findings. Neovascular growth will be        semi quantitatively scored. Image analysis is also available.

Example 8B Thyroid Hormone and Diabetic Retinopathy

A protocol disclosed in J de la Cruz et al., J Pharmacol Exp Ther280:454-459, 1997, is used for the administration of tetrac, tetracnanoparticles and polymers conjugated via a covalent bond using an esterlinkage to rats that have streptozotocin (STZ)-induced experimentaldiabetes and diabetic retinopathy. The results of this experimentdemonstrated the inhibition by tetrac of the appearance of proliferativeretinopathy (angiogenesis) and reduction in neovascularization,producing similar results similar to example 13 described belowincluding 50% inhibition of neovascularization. Tetrac inhibition ofretinal neovascularization in the oxygen-induced retinopathy model,makes tetrac a viable therapeutic strategy for proliferative diabeticretinopathy.

Example 9 In Vitro Human Epithelial and Fibroblast Wound Healing

The in vitro 2-dimensional wound healing method is as described inMohamed S, Nadijcka D, Hanson, V. Wound healing properties of cimetidinein vitro. Drug Intell Clin Pharm 20: 973-975; 1986, incorporated hereinby reference in its entirety. Additionally, a 3-dimensional woundhealing method already established in our Laboratory will be utilized inthis study (see below). Data show potent stimulation of wound healing bythyroid hormone.

In Vitro 3D Wound Healing Assay of Human Dermal Fibroblast Cells: Step1: Prepare Contracted Collagen Gels:

-   -   1) Coat 24-well plate with 350 ul 2% BSA at RT for 2 hr,    -   2) 80% confluent NHDF (normal human dermal fibroblast cells,        Passage 5-9) are trypsinized and neutralized with growth medium,        centrifuge and wash once with PBS    -   3) Prepare collagen-cell mixture, mix gently and always on ice:

Stock solution Final Concentration 5xDMEC 1xDMEM 3 mg/ml vitrogen 2mg/ml ddH2O optimal NHDF 2 × 10~5 cells/ml FBS 1%

-   -   4) Aspire 2% BSA from 24 well plate, add collagen-cell mixture        350 μl/well, and incubate the plate in 37° C. CO2 incubator.    -   5) After 1 hr, add DMEM+5% FBS medium 0.5 ml/well, use a 10 ul        tip Detach the collagen gel from the edge of each well, then        incubate for 2 days. The fibroblast cells will contract the        collagen gel

Step 2: Prepare 3D Fibrin Wound Clot and Embed Wounded Collagen Culture

-   -   1) Prepare fibrinogen solution (1 mg/ml) with or without testing        regents. 350 ul fibrinogen solution for each well in eppendorf        tube.

Stock solution Final Concentration 5xDMEC 1xDMEM Fibrinogen 1 mg/mlddH2O optimal testing regents optimal concentration FBS 1% or 5%

-   -   2) Cut each contracted collagen gel from middle with scissors.        Wash the gel with PBS and transfer the gel to the center of each        well of 24 well plate    -   3) Add 1.5 μl of human thrombin (0.25 U/μl) to each tube, mix        well and then add the solution around the collagen gel, the        solution will polymerize in 10 mins.

After 20 mins, add DMEM+1% (or 5%) FBS with or without testing agent,450 μl/well and incubate the plate in 37° C. CO2 incubator for up to 5days. Take pictures on each day.

In Vivo Wound Healing in Diabetic Rats:

Using an acute incision wound model in diabetic rats, the effects ofthyroid hormone analogs and its conjugated forms are tested. The rate ofwound closure, breaking strength analyses and histology are performedperiodically on days 3-21.

Example 10 Rodent Model of Myocardial Infarction

The coronary artery ligation model of myocardial infarction is used toinvestigate cardiac function in rats. The rat is initially anesthetizedwith xylazine and ketamine, and after appropriate anesthesia isobtained, the trachea is intubated and positive pressure ventilation isinitiated. The animal is placed supine with its extremities looselytaped and a median sternotomy is performed. The heart is gentlyexteriorized and a 6-O suture is firmly tied around the left anteriordescending coronary artery. The heart is rapidly replaced in the chestand the thoracotomy incision is closed with a 3-O purse string suturefollowed by skin closure with interrupted sutures or surgical clips.Animals are placed on a temperature regulated heating pad and closelyobserved during recovery. Supplemental oxygen and cardiopulmonaryresuscitation are administered if necessary. After recovery, the rat isreturned to the animal care facility. Such coronary artery ligation inthe rat produces large anterior wall myocardial infarctions. The 48 hr.mortality for this procedure can be as high as 50%, and there isvariability in the size of the infarct produced by this procedure. Basedon these considerations, and prior experience, to obtain 16-20 rats withlarge infarcts so that the two models of thyroid hormone deliverydiscussed below can be compared, approximately 400 rats are required.

These experiments are designed to show that systemic administration ofthyroid hormone either before or after coronary artery ligation leads tobeneficial effects in intact animals, including the extent ofhemodynamic abnormalities assessed by echocardiography and hemodynamicmeasurements, and reduction of infarct size. Outcome measurements areproposed at three weeks post-infarction. Although some rats may have noinfarction, or only a small infarction is produced, these rats can beidentified by normal echocardiograms and normal hemodynamics (LVend-diastolic pressure <8 mm Hg).

Thyroid Hormone Delivery

There are two delivery approaches. In the first, the thyroid hormoneconjugated to a nanoparticle may be directly injected into theperi-infarct myocardium. As the demarcation between normal and ischemicmyocardium is easily identified during the acute open chest occlusion,this approach provides sufficient delivery of hormone to detectangiogenic effects.

Although the first model is useful in patients undergoing coronaryartery bypass surgery, and constitutes proof of principle that one localinjection induces angiogenesis, a broader approach using a second modelcan also be used. In the second model, a catheter retrograde is placedinto the left ventricle via a carotid artery in the anesthetized ratprior to inducing myocardial infarction. Alternatively, a direct needlepuncture of the aorta, just above the aortic valve, is performed. Theintracoronary injection of the thyroid hormone conjugated to a polymeris then simulated by abruptly occluding the aorta above the origin ofthe coronary vessels for several seconds, thereby producing isovolumiccontractions. The conjugated thyroid hormone is then injected into theleft ventricle or aorta immediately after aortic constriction. Theresulting isovolumic contractions propel blood down the coronary vesselsperfusing the entire myocardium with thyroid hormone. This procedure canbe done as many times as necessary to achieve effectiveness. The numberof injections depends on the doses used and the formation of new bloodvessels.

Echocardiography:

A method for obtaining 2-D and M-mode echocardiograms in unanesthetizedrats has been developed. Left ventricular dimensions, function, wallthickness and wall motion can be reproducibly and reliably measured. Themeasurements are carried out in a blinded fashion to eliminate bias withrespect to thyroid hormone administration.

Hemodynamics:

Hemodynamic measurements are used to determine the degree of leftventricular impairment. Rats are anesthetized with isoflurane. Throughan incision along the right anterior neck, the right carotid artery andthe right jugular vein are isolated and cannulated with a pressuretransducing catheter (Millar, SPR-612, 1.2 Fr). The followingmeasurements are then made: heart rate, systolic and diastolic BP, meanarterial pressure, left ventricular systolic and end-diastolic pressure,and + and −dP/dt. Of particular utility are measurements of leftventricular end-diastolic pressure, progressive elevation of whichcorrelates with the degree of myocardial damage.

Infarct Size:

Rats are sacrificed for measurement of infarct size using TTCmethodology.

Morphometry

Microvessel density [microvessels/mm²] will be measured in the infarctarea, peri-infarct area, and in the spared myocardium opposing theinfarction, usually the posterior wall. From each rat, 7-10 microscopichigh power fields [×400] with transversely sectioned myocytes will bedigitally recorded using Image Analysis software. Microvessels will becounted by a blinded investigator. The microcirculation will be definedas vessels beyond third order arterioles with a diameter of 150micrometers or less, supplying tissue between arterioles and venules. Tocorrect for differences in left ventricular hypertrophy, microvesseldensity will be divided by LV weight corrected for body weight.Myocardium from sham operated rats will serves as controls.

Example 11 Effects of the αvβ3 Antagonists on the Pro-AngiogenesisEffect of T4 or FGF2

The αvβ3 inhibitor LM609 totally inhibited both FGF2 and T4-inducedpro-angiogenic effects in the CAM model at 10 micrograms (FIG. 16).

Example 12 Inhibition of Cancer-Related New Blood Vessel Growth

A protocol disclosed in J. Bennett, Proc Natl Acad Sci USA 99:2211-2215,2002, is used for the administration of tetraiodothyroacetic (tetrac) toSCID mice that have received implants of human breast cancer cells(MCF-7). Tetrac is provided in drinking water to raise the circulatinglevel of the hormone analog in the mouse model to 10-6 M. The endpointwas the inhibitory action of tetrac on angiogenesis of the implantedprimary and metastatic tumors.

Example 13 Efficacy of Tetrac in Retinal Neovascularization in Mice withRetinopathy of Prematurity

Retinal angiogenesis is a major cause of blindness in ischemicretinopathies including diabetic retinopathy and retinopathy ofprematurity. The mice model used was the Oxygen-induced retinopathymouse model as described by Smith et al. (Smith LE 1994). As depicted inFIG. 21, the neonatal mice were exposed to hyperoxic conditions for 5days, beginning at age p7 for the pups and continuing through p12 whenthey are returned to normal oxygen atmosphere (8-10 animals/group).Three groups were used, the control consisting of treatment with PBS,treatment group consisting of tetrac and a treatment group consisting oftetrac nanoparticles (TNP). Tetrac and TNP (consisting of tetrac PLGAconjugated Nanoparticles via an ester linkage) dosed at 1 mg/kg in PBSat pH 7-7.5, and the Control (PBS) were administered on postnatal day 12and 15. While PLGA was used, similar results are expected if otherpolymers were substituted such as polyglycolide, or polylactic acid. Theadministration was conducted intraperitoneally (IP). The nanoformulationenables for longer residence time on the cornea allowing for greaterpermeation of tetrac or its tetrac PLGA conjugated Nanoparticles.

On day 17 the animals were scarified and eyes were removed for retinalneovascularization evaluation as summarized in FIG. 21. The eyes wereremoved fixed in formalin and were stained with H&E stain. As evidencedby the immunohistochemical staining of ROP mice in FIG. 21, normalvessels were present in the eye at room temperature, while capillarydrop out occurred between p7 and p12 due to a decrease in VEGF and otherangiogenic factors when the O₂ were at 75±2%. When the mice were placedback in normal room air, neovascularization is observed due to anincrease in VEGF and other angiogenic factors. FIG. 23 a depicts thecomparison between the effects of exposure to room air versus 75% O₂ onthe vascularization area of murine retinas. This may be compared withFIG. 23 b which depicts the effects of tetrac and tetrac nanoparticleson the vascularization of murine retinas.

FIG. 22 depicts data representing and comparing the mean total area ofneovascularization after administration of tetrac at 10 mg/kg, tetracnanoparticles at 1 mg/kg and the control on day 12 and 15. As seen fromthe depicted data, the administration of either tetrac ornanoparticulate tetrac resulted in 50% inhibition of neovascularization.The resulting effects are comparable with potent αVβ3 antagonist, XT199.Tetrac and nano-tetrac inhibition of retinal neovascularization in theoxygen-induced retinopathy model makes tetrac a viable therapeuticstrategy for proliferative diabetic retinopathy.

Example 14 Nano-Tetrac Exhibiting Both Thyroid Hormone Dependent andIndependent Anti-Angiogenic Effects

Nanoparticulate tetrac is capable of binding to the αVβ3 receptor onactively dividing endothelial cells. We tested its effects onneovascularization using the chicken chorioallantoic membrane (CAM)model to determine the effectiveness of tetrac and nanoparticulatetetrac conjugated to polymers as described above, in treatingneovascularization that occurs from diabetic retinopathy. 10-day-oldchick embryos were purchased and incubated at 37° C. with 55% relativehumidity. A hypodermic needle was used to make a small hole in theeggshell at the air sac, and a second hole was made on the long side ofthe egg, directly over an avascular portion of the embryonic membraneidentified by candling. A false air sac was created beneath the secondhole by distal application of negative pressure to separate the CAM fromthe shell. A ˜1.0 cm² window was cut in the shell over the dropped CAM,allowing direct access to the underlying membrane. Filter disks thatwere pre-incubated with FGF2 (1 μg/ml in PBS) were placed on CAMs onday 1. Thirty minutes after filter placement, 2 μg of either tetrac orNT were added to the filter and the eggs were incubated at 37° C. After3 days, the CAM tissue directly beneath each filter disk was resected,washed three times with PBS, placed in 35 mm petri dishes, and examinedunder SV6 stereo-microscope (Carl Zeiss, NY) at 50×. Digital images ofCAM sections beneath the filters were collected using a 3-charge-coupleddevice color video camera system (Toshiba America, NY), and the numberof branch points in the blood vessels were analyzed with Image-Prosoftware (Media Cybernetics, MD). The number of branch points in acircular region equal to the area of filter disk were counted. Thephotomicrographs comparing the CAMS as well as the results are depictedin FIGS. 24 a and 24 b. Eight CAM preparations were analyzed for eachtreatment. Each experiment was performed three times, and this procedurewas repeated with other related factors and compounds known to promoteangiogenesis.

The results show that angiogenesis promoted by T3 and T4 is blocked bynanoparticulate tetrac, which directly blocks T3 and T4 binding to theαVβ3 receptor decreased neovascularization. In addition, independent ofthyroid hormone, nanoparticulate tetrac and tetrac, conjugated topolymers also effectively blocks angiogenesis promoted by TNF-α and bFGFvia the αVβ3 receptor. Angiogenesis mediated via VEGF is also inhibitedby nanoparticulate tetrac, suggesting that nanoparticulate tetrac blocksangiogenesis promoted synergistically by VEGF and the α_(v)β3 receptor.Nanoparticulate tetrac also effectively inhibits angiogenesis promotedby bradykinin and angiotensin via FGF2 and VEGFR2, respectively.Additionally, nanoparticulate tetrac inhibits LPS, which promotesangiogenesis via TNF receptor-associated factor 6 (TRAF6)-mediatedactivation of NFκB and c-Jun N-terminal kinase. Nanoparticulate tetracthus effectively blocks angiogenesis triggered via multifactorialpathways, a vital quality for a tumor angiogenesis inhibitor, as primaryand metastatic tumors utilize multiple angiogenesis promoting factorsfor their growth.

Example 15 CAM Tumor Growth Studies

For the tumor angiogenesis and tumor growth studies of primary andmetastatic tumors, LNCaP or PC3 cells were introduced topically into theCAM. Test compounds were added to the cancer cells in matrigel to assesstheir ability to target the tumor or tumor vasculature. Tumors wereexcised, and examined under a stereomicroscope at 50-× magnification.Digital images of the tumor were collected using a 3-CCD color videocamera system and analyzed with Image-Pro Plus software as depicted inFIG. 25. The numbers of vessel branch points were counted for eachsection. Portions of the tumor were extracted for hemoglobindeterminations. Studies were performed to evaluate the efficiency andtiming of targeted nanoparticles localization into the tumor vasculatureusing green or red fluorescence-labeled nanoparticles. For example,Alexa-488 labeled nanoparticles were used along withAlexa-543/594-labeled tetrac. Individual CAMs were harvested at varioustime points after administration of the nanoparticles and the number offluorescent particles quantified in either tumor vasculature or tumortissue.

Shown here is an example of LNCaP tumor grown in the CAM. As depicted inFIG. 25, the tumor's 2501 extensive angiogenesis apparent. In theresults shown below in Table 3, LNCaP tumors were grown in the tumorimplant model. CAMs were treated with PBS (control), tetrac ornanoparticulate tetrac conjugated to polymers via a covalent bond. Atthe end of the experiment, tumors were removed and weighed, thenextracted for measurement of hemoglobin to evaluate the amount ofangiogenesis.

Tumor tissue derived from the experiments shown in Table 3 was processedto evaluate angiogenesis by extraction of the tissue and measuringhemoglobin content using Drabkin's reagent. This method correlates wellwith branch point analysis data (Table 3), showing a similar extent ofinhibition of tumor-induced angiogenesis. Data demonstrates that bothtetrac and Nanoparticulate tetrac effectively inhibit the growth ofthese primary and metastatic tumors in the CAM Tumor Implant model.Further, both agents significantly inhibited tumor-induced angiogenesis(FIG. 26), a key factor in metastasis and tumor growth.

TABLE 3 Tetrac and Tetrac-nano inhibit LNCaP Tumor growth and Tumor-induced Angiogenesis in the CAM Tumor Implant Model Tumor weight ±Branch CAM Conditions SEM (mg) Points ± SEM LNCaP (0.5 × 10⁹ cells/CAM)35.3 ± 8.1   93.3 ± 8   LNCaP + Tetrac (10 ug/CAM) 13 ± 3.1* 59 ± 5.2*LNCaP + Tetrac - nano (1 ug/CAM) 21 ± 4.4* 55 ± 3.6* Data represent mean± SEM, n = 8 per group. Either tetrac or Nanoparticulate tetrac resultedin significant suppression of tumor growth or tumor-mediatedangiogenesis as compared to untreated LNCaP cancer cells, *P < 0.01.There were no statistically significant differences between tetrac andNanoparticulate tetrac.

Example 16 Effect of Tetrac on the Proliferation of Drug ResistantTumors In Vivo

Our in vitro studies demonstrated that tetrac reversed drug resistancein several tumor cell lines. To investigate the in vivo relevance oftetrac in suppressing drug resistance, we tested its effect, eitheralone or in combination with doxorubicin, in nude mice bearing xenograftof drug resistant cancer cells. While doxorubicin was used in thisexample, doxorubicin could have been substituted for any of thechemotherapeutic agents described above including etoposide,cyclophosphamide, 5-fluorouracil, cisplatin, trichostatin A, paclitaxel,gemcitabine, taxotere or cisplatinum.

Mice were injected with the doxorubicin-resistant breast cancer cellline MCF7/R and when the tumors became palpable, they received threedrug injections of doxorubicin alone, tetrac alone, or the combinationof both. The maximal tolerated dose (MTD) for doxorubicin was 2.5 mg/Kg,however, in the case of tetrac; no toxicity was detected for up to 60mg/ml. The data, which is depicted in FIG. 27, indicates thatdoxorubicin alone (2 mg/Kg) had no noticeable effect on tumor growth. Incontrast, tetrac at 30 mg/ml alone reduced tumor growth by about 70%.This effect was not further improved by the combination of both drugssuggesting a lack of synergistic effect at the concentrations used.Interestingly, the drug concentration of tetrac was well tolerated andno significant weight loss was noticed in the treated animals during theexperiments. These findings suggest that tetrac is able to suppress theproliferation of drug resistant tumors in vivo and thus may be capableof treating drug resistant tumors.

Example 17 Anti-Tumor Activity of Tetrac and Tetrac-PLGA Nanoparticlesin LNCaP Mice Xenograft

Mice were xenografted with 10 million LNCaP cells implanted in matrigel.The mice were treated daily with either tetrac or tetrac-PLGA conjugatedpolymer nanoparticles. The mice were administered via intraperitonealinjection. The results, as depicted in FIGS. 28 a and 28 b demonstratethe effectiveness of tetrac and tetrac-PLGA nanoparticles in reducingtumor growth of primary and metastatic tumors in the mice xenografts andthe effects on tumor angiogenesis respectively.

Example 18 Suppression of Prostate Tumor Growth Compared with Paclitaxel

Tumor fragments (1 mm³) were prepared from prostate tumors growingsubcutaneously in nude mice. Either Lucefrin transfected prostate cancercell lines (1 million cells) or tumor fragments were implanted bysurgical orthotopic implantation in the lateral lobe of the prostate,which was exposed after a lower midline abdominal incision. After properexposure of the bladder and prostate, the capsule of the prostate wasopened and the two tumor fragments (1 mm³) were inserted into thecapsule. The capsule was then closed with an 8-0 surgical suture. Theincision in the abdominal wall was closed with a 6-0 surgical suture inone layer. The animals were kept under isoflurane anesthesia duringsurgery. All procedures of the operation described above were performedusing a surgical microscope.

Male nude mice were implanted orthotopically with prostate cancer celltransfected with lucefrin and animals were randomized into 3 differentarms as shown below:

1. PBS 1 ml/kg, IP daily (n=4)

2. Paclitaxel 0.3 mg/kg, IP daily (n=5)

3. Tetrac 1 mg/kg, IP daily (n=5)

As depicted in FIG. 29, the tetrac administered intraperitoneally at 1mg/kg daily for 17 days resulted in distinct suppression of prostatetumor growth comparable to the reduction obtained by the cytotoxic,chemotherapeutic agent, paclitaxel.

Example 19 Nanoparticulate Tetrac Exerts Anti-Proliferative Effects onPancreatic Adenocarcinoma Cells

In this study, we used any two of the following: Panc-1, MiaPaCa-2 andAsPc-1 PDAC cell lines, as they all express significant levels of αVβ3.Cells were counted and plated in media with either tetrac ornanoparticulate tetrac (10 μM). Media were replenished daily with freshtetrac and nanoparticulate tetrac (NT-1 or NT-2 batches). Viable cellswere counted by trypan blue exclusion assays. Cells were cultured for 8days in the presence of the vehicle (control), tetrac andnanoparticulate tetrac. As depicted in FIG. 30, cells were counted ondays 3, 6 and 8. By day 8, the Panc-1 cell counts (data not shown forAsPc-1 cells) were reduced by approximately 20% with tetrac treatment(*P<0.04) and by 35-40% with nanoparticulate tetrac treatment(**P<0.01), compared to untreated cells. Both unmodified tetrac andnanoparticulate tetrac effectively reduced proliferation in Panc-1cells. Nanoparticulate tetrac was show to be more effective than tetrac.

Example 20 Nanoparticulate Tetrac Regulates Gene Expression to InhibitCell Survival, Cell Proliferation and Angiogenesis

Using microarray analysis, we found that both tetrac and nanoparticulatetetrac alter the expression of genes relevant to cell division andangiogenesis in human breast cancer cells and in medullary thyroidcarcinoma cells. We used western analysis to test the expression of thepro-apoptotic protein, bcl-Xs, and the anti-angiogenic proteinthrombospondin (THBS1), in AsPc-1 and Panc-1. We also used RT-PCR toanalyze expression of the mRNAs for the cell cycle regulatory genes p21and p53. To do this, cells were plated, treated with either tetrac ornanoparticulate tetrac. Total cell extracts were probed withanti-bcl-Xs, anti-THBS1, and anti-β-actin (loading control) antibodies.Tetrac and nanoparticulate tetrac both increased bcl-Xs and THBS1protein expression by 2-fold and 4-fold, respectively, compared to theuntreated controls as depicted in FIG. 31 a. These results suggest thatthe anti-angiogenic and anti-survival effects of nanoparticulate tetracand tetrac occur via modulation of gene expression. Data with AsPc-1,p21 and p53 (GAPDH, internal controls) mRNA levels were significantlydecreased by both tetrac and nanoparticulate tetrac, whilenanoparticulate tetrac alone significantly reduced EGFR expressionlevels (as depicted in FIG. 31 b and FIG. 31 c), which collectivelycould slow cell cycle progression. Thus, nanotetrac affects cellproliferation, cell survival, and angiogenesis by modulating geneexpression.

Example 21 Tetrac Exerts Anti-Tumorigenic and Anti-Angiogenic Effects onPancreatic Xenograft

To complement our in vitro observations, we tested the effects of tetracand nanoparticulate tetrac conjugated to polymers via a covalent bond onprimary and metastatic tumor growth, and tumor angiogenesis in vivo.Female nude mice were purchased at age 5-6 weeks (20 g body weight) andhoused in the animal facility. The animal protocol was approved by theInstitutional Animal Care and Use Committee (IACUC). Approximately 2×10⁶AsPc-1 cells in 100 μl of growth medium were mixed with 100 μl ofMatrigel® and injected subcutaneously into the left and right flanks (2per side) of 18 animals. Tumors (4 per animal) were measured daily withcalipers, and tumor volumes were calculated using the formula, W×L²/2,where W is width and L is length. When the tumor volumes reached ˜250mm³, the animals were randomized into 3 groups (n=6/group) andsubcutaneously injected with solvent (control), tetrac (10 mg/kg bodyweight), or nanoparticulate tetrac (1 mg/kg body weight). Tumor volumeswere measured daily for the next 15 days. As shown in FIG. 32 a-c,untreated animal tumor volumes continued to increase until the 15^(th)day of treatment. Treatment with either tetrac or nanoparticulate tetracresulted in progressive reductions of the tumor volumes of AsPc-1xenograft, which reached statistical significance by treatment days 5-6(P<0.05). By the end of day 15 of treatment, both agents decreased thetumor volume to that of the original implant. On the last day of tumormeasurement, the animals were observed by IVIS imaging as depicted inFIG. 32 b, which revealed that the reductions in tumor mass correlatedwith reductions in the number of viable cells, indicated by reducedluciferase signal intensities. The animals were humanely sacrificed andtheir tumors were harvested.

The hemoglobin contents of the tumor masses were measured to obtain anindirect estimate of vascularity. For this purpose, each tumor washomogenized in double-distilled water, centrifuged (4,000×g for 10 min),and the supernatants were collected. 50 μl of each supernatant was mixedwith 50 μl of Drabkin's reagent, incubated at room temperature for 30min, transferred to 96-well plates, and absorbances were measured at 540nm using a micro-plate reader. The hemoglobin concentration (mg/mL) wasestimated from a standard curve. To establish the use of tumorhemoglobin content as a measure for tumor neovascularization, wevalidated a linear correlation between tumor hemoglobin content with thedegree of tumor neovascularization using anti-CD31 staining. As shown inFIG. 32 c, the hemoglobin contents of the xenograft (an index of tumorangiogenesis) collectively decreased by 60% in animals treated for 15days with tetrac or tetrac nanoparticles (*P<0.05), compared with tumorsin untreated control animals. Thus, both tetrac and NT at ten-fold lowerdoses significantly decreased tumor growth and tumor angiogenesis in anin vivo xenograft pancreatic tumor model.

Example 22 Nanoparticulate Tetrac Sensitizes Drug-Resistant PancreaticCells to Gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) patients have poor survivalrates, in part due to increased resistance to chemotherapy. Previousstudies suggest that the αVβ3 receptor promotes drug resistance incancer cells and that non-genomic actions of thyroid hormone (TH)regulate the expression of genes that are relevant to drug resistancevia αVβ3 receptor. This led us to hypothesize that nanoparticulatetetrac reverses drug resistance in pancreatic cells expressing the αVβ3receptor by binding to the receptor and blocking both TH-dependent andTH-independent downstream pathways. We used MiaPaCa-2 cells that expressαVβ3 receptor and are resistant to gemcitabine. Equal numbers of cellswere plated and treated with nanoparticulate tetrac alone (5 μg/ml),gemcitabine alone (1 μM) or nanoparticulate tetrac with gemcitabine.After 36 hours, cell survival was measured using an Apo-direct kit,which labels DNA breaks so that apoptotic cells can be detected by flowcytometry. As depicted in FIG. 33, we found that gemcitabine treatmentalone resulted in death of 5% of the cells, while nanoparticulate tetractreatment alone resulted in 10% cell death. However, nanoparticulatetetrac in combination with gemcitabine increased the cell death to 15%.This result suggests that nanoparticulate tetrac treatment causedgemcitabine-resistant cells to become susceptible to gemcitabinetreatment. Thus, nanoparticulate tetrac treatment of chemo-resistantPDAC cells expressing the αVβ3 receptor resulted in the cells becomingchemo-sensitive.

Example 23 Nanoparticulate Tetrac Regulates the Expression of miRNA-15a

Nanoparticulate tetrac may mediate a wide array of anti-canceractivities via αVβ3 receptor that are both TH-dependent andTH-independent. MiRNAs play key roles in controlling the expression ofmany cellular proteins, enabling them to regulate many cellular pathwaysand thus any deregulation in their physiological levels may largelycontribute to diseases. Recent evidence showed that T3 hormone increasedmiR-350 expression in cardiomyocytes, which in turn stabilized thetranscripts of angiotensin II type 1 receptor (AT1R) gene to increaseits translational efficiency. This suggests that TH can mediate geneexpression by regulating miRNA levels. We hypothesized thatnanoparticulate tetrac, which blocks the effects of T3 and T4 at αVβ3receptor may also be able to regulate miRNAs. Emerging evidence showsthat alterations of miRNA expression, such as miR-21, miR-10b andmiR-15a play a significant role in tumorigenesis. We thereforeinvestigated if nanoparticulate tetrac treatment regulated theexpression of miR-15a, which is also shown to regulate cellproliferation, angiogenesis and chemoresistance in various cancer types.

In PDAC, the levels of miR-15a are down regulated and overexpression ofexogenous miR-15a inhibited the viability of pancreatic cancer cells. Wetherefore determined whether nanoparticulate tetrac up-regulates miR-15aexpression in PDAC cells. Equal numbers of AsPc-1 cells were seeded andtreated with nanoparticulate tetrac, for 16 hours. Enriched miRNAs wereisolated from the cells, using miRNeasy kit, reverse transcribed andqPCR was performed to amplify miR-15a and RNU6 miRNA, an internalcontrol, in triplicate. The expression of miR-15a in nanoparticulatetetrac treated sample relative to that in untreated was determined to be1.4 fold by calculating relative quantification (RQ), which is the foldchange compared to the calibrator (RNU6), as shown in Table 4 below.Thus, nanoparticulate tetrac treatment increases the miR-15a levels by1.4 fold compared to untreated sample with 95% confidence interval. Weexpect the fold-difference to increase, with longer treatment time.

TABLE 4 Relative Quantification (RQ) of miR-15a in AsPc-1 cells. SampleRQ 95% Conf. Int. Untreated 1 0.918-1.089 NT 1.379 1.353-1.406

Example 24 Effects of Tetrac and Nanotetrac on Non-Small Cell LungCancer

Cells and cell culture: Human non-small cell lung cancer (NSCLC)NCI-H1299 cells were purchased from American Type Culture Collection(Manassas, Va.) and cultured as instructed by the supplier, usingcomplete growth RPMI medium supplemented with 10% FBS. Cells werecultured in a 5% CO2/air atmosphere at 37° C. to sub-confluence and thentreated with 0.25% (w/v) trypsin/EDTA to affect cell release from theculture vessel. After cells were washed with culture medium, they weresuspended in DMEM that was free of phenol red and FBS and counted.

We studied the inhibitory effect of tetrac and tetrac NP xenograftgrowth in nude mice. Xenografts of 4×106 H1299 cells were implanted andanimals were treated IP with unmodified tetrac (1 mg/kg body weight) ortetrac-NP (1 mg tetrac as the nanoparticle/kg) every 2 d, beginning onday 10, when tumor volume was 200-300 mm³. A factor contributing toincrease in tumor volume is assumed to be endogenous thyroid hormone.Treatment with either tetrac or tetrac-NP resulted within 2-3 d inreduction of tumor volume (FIG. 34) and for the 20-d duration oftreatment the volume rose negligibly above that at day zero (FIG. 34).At the end of the study, tumor mass was directly measured and, comparedto controls, both agents resulted in significant (p<0.01) reduction ofmass (FIG. 35A). In FIG. 35B, tumor hemoglobin (Hgb) content, an indexof vascularity/angiogenesis, is shown to be reduced by both tetracformulations. Animal body weight was unaffected by tetrac and tetrac NP.

Additional studies were conducted of the effectiveness of tetrac andtetrac-NP against larger tumor implants. Implants of 107 NCI-H1299 cells(10-fold the implant size shown in FIGS. 35-36) resulted in greaterxenograft growth in control animals. Daily treatment of animals withtetrac (2.0 mg/kg, IP) or tetrac-NP (1.86 mg tetrac as thenanoparticle/kg, IP) resulted in suppression in tumor growth within 2-3d (FIG. 36A). This difference in growth rate persisted throughout the20-d duration of the study. Both agents resulted in significant (p<0.01)inhibition of tumor mass, compared to control (FIG. 36B).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method for treating a condition amenable to theinhibition of angiogenesis comprising the steps of: administering to asubject in need thereof an effective amount of a compound selected fromthe group consisting of tetriodothyroacetic acid (tetrac),triiodothyroacetic acid (triac) and a combination thereof conjugated viaa covalent bond to a polymer, wherein said polymer is polyglycolide,polylactic acid, or co-polymers thereof, wherein said polymer isformulated into a nanoparticle, wherein said nanoparticle is less than200 nanometers, and wherein the administered compound acts at the cellmembrane level to inhibit pro-angiogenesis agents.
 2. The method ofclaim 1, wherein the condition amenable to treatment byanti-angiogenesis is selected from the group consisting of a primarytumor, metastatic tumor and diabetic retinopathy.
 3. The method of claim1, wherein the covalent bond is selected from a group consisting of anester linkage, ether linkage, sulfhydryl linkage, and an anhydridelinkage.
 4. The method of claim 1, wherein the step of administeringincludes a route of administration selected from the group consisting ofparenteral, oral, rectal, topical, intratumoral, intraocular, andcombinations thereof.
 5. The method according to claim 1, wherein thecompound is co-administered with at least one chemotherapeutic agent. 6.The method according to claim 5, wherein the at least onechemotherapeutic agent is selected from a group consisting ofdoxorubicin, etoposide, cyclophophamide, 5-fluoracil, cisplatin,trichostatin A, paclitaxel, gemcitabine, taxotere, cisplatinum,carboplatinum, irinotecan, topotecan, adrimycin, bortezomib,combinations thereof and derivatives thereof.